Articles comprising a silk polypeptide for antigen delivery

ABSTRACT

The present invention relates to a polypeptide comprising a silk polypeptide and an antigen. Further, the present invention relates to an article comprising the polypeptide. Furthermore, the present invention relates to a pharmaceutical composition comprising the article. In addition, the present invention relates to the article or pharmaceutical composition for use as a pharmaceutical, for inducing an immune response and/or for use in a prophylactic and/or therapeutic treatment of a disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/307,622, filed Dec. 6, 2018, which is a 371 National StageApplication of PCT/EP2017/064436, International Filing Date Jun. 13,2017, which claims priority to EP 16175724.0 filed Jun. 22, 2016, thedisclosures of which are incorporated herein by reference for allpurposes.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file Sequence_Listing_1116752.txtcreated on Dec. 5, 2018, 32,768 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

The present invention relates to a polypeptide comprising a silkpolypeptide and an antigen. Further, the present invention relates to anarticle comprising the polypeptide. Furthermore, the present inventionrelates to a pharmaceutical composition comprising the article. Inaddition, the present invention relates to the article or pharmaceuticalcomposition for use as a pharmaceutical, for inducing an immune responseand/or for use in a prophylactic and/or therapeutic treatment of adisease.

BACKGROUND OF THE INVENTION

Antigen delivery into animal cells, e.g. human cells, is important inthe medical field. It has, for example, high potential in the commercialarea of vaccination. It is, in addition, highly relevant with respect tothe improvement of therapeutic approaches for treating animals, e.g.humans. Cancer, autoimmune diseases, mycosis, infections with virusesand bacteria or therapeutic vaccination against Hepatitis B/C or HIVare, for example, promising targets for immunotherapeutic treatments.

Immunization by vaccination is one of the most productive tools inmodern medical practice. The body's ability to protect itself fromforeign pathogens relies on its ability to recognize and react toinfectious materials once they are presented to the host immune cells,in a process known as the adaptive immune response. Currently employedcompositions for immunization deliver antigens with a mixture ofstabilizers, preservatives, and adjuvants. The antigen is typically aprotein component of a bacterium or virus. The vaccine comprising theantigen induces both humoral and cellular responses to said antigen. Theeffectiveness of such vaccination largely depends on the antigen'simmunogenicity, which is in turn a function of the antigen's size,molecular complexity, degree of “foreignness” and capacity to be cleavedinto peptides by antigen-presenting cells (APCs). The antigen-stimulatedand activated APCs migrate to draining lymph nodes (DLNs), where theantigen is then presented to naïve T helper cells. Subsequently theantigen is presented to B cells resulting in systemic antibodyproduction as well as priming of memory B cells. The initial productionof antibodies begins to decline approximately three weeks post-primaryexposure, but can be further enhanced via a second contact with the sameantigen. Therefore, booster shots are strongly advocated and generallyinduce high concentrations of antigen-specific antibodies. Dendriticcells (DCs) as specialized antigen-presenting cells (APCs) play criticalroles in both innate and adaptive immunity. DCs are specializedantigen-presenting cells with the unique capability to capture andprocess antigens, migrate from the periphery to a lymphoid organ, andpresent the antigens to resting T cells in a major histocompatibilitycomplex (MHC)-restricted fashion.

However, one of the drawbacks of the currently available antigendelivery material is the cytotoxic effect this material has on targetcells. Given the potential therapeutic and clinical uses of the deliverymaterial, less cytotoxic material is needed to carry antigens intovarious cells without affecting inherent signaling pathways and systems.In addition, vaccines comprising antigens are often not sufficientlyabsorbed by the cells/internalized into the cells. Thus, there is a needfor a non-cytotoxic article comprising an antigen having a structurewhich is preferentially absorbed by the cells/internalized into thecells. Moreover, artificial antigen carrier vehicle which are composedof a single chain polypeptide comprising a carrier polypeptide and anantigen are not known in the art.

The inventors of the present patent application produced for the firsttime antigen carrier articles which are composed of a single chainpolypeptide comprising a silk polypeptide as carrier polypeptide as wellas an antigen. Further, the inventors of the present patent applicationsurprisingly found that antigens which are part of articles, e.g.particles, comprising silk polypeptides are preferentially taken up byanimal cells, e.g. human cells. In this context, the inventors of thepresent patent application were surprised that the articles, e.g.particles, which are mainly composed of silk, function as an auxiliarymaterial which stabilizes the antigen and makes it transportable. Thearticles, e.g. particles, can be degraded within the organism (e.g.human body) without traces. Furthermore, the inventors of the presentpatent application surprisingly found that the use of adjuvants in apharmaceutical composition comprising antigens which are part of thearticles, e.g. particles, comprising silk polypeptides is no longerrequired. The effectiveness of an antigen comprised in a compositionwithout adjuvants is at least as good as or comparable to the sameantigen comprised in a composition with adjuvants. The omission ofadjuvants has the positive effect that side effects and vaccineincompatibility can be reduced, in some cases even avoided. In addition,the articles, e.g. particles, comprising silk polypeptides and antigensare non-toxic, in particular non-cytotoxic, and non-immunogenic. Thearticles, e.g. particles, can be readily manufactured in an one-stepproduction process without the need of organic solvents or toxicsubstances. Additionally, a one-step production process reduces therisks of harmful impurities or contaminations and has significantadvantages regarding documentation and regulatory aspects. A one-stepproduction process means that no additional loading step is necessaryafter article formation. The one-step production process is particularlydesirable as it is easily up-scalable to manufacturing scale.Furthermore the antigen is exactly defined by the genetic code in thepolypeptide and can, therefore, not be degraded in the course of theotherwise needed loading procedure.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a polypeptidecomprising

-   (i) a silk polypeptide and-   (ii) an antigen.

In a second aspect, the present invention relates to a nucleic acidmolecule encoding the polypeptide of the first aspect.

In a third aspect, the present invention relates to a method forproducing a polypeptide comprising the step of:

-   (a) expressing the nucleic acid molecule of the second aspect in a    cell, thereby producing the polypeptide in the cell.

In a fourth aspect, the present invention relates to an articlecomprising the polypeptide of the first aspect.

In a fifth aspect, the present invention relates to a method forproducing an article comprising the steps of:

-   (a) providing an aqueous solution comprising the polypeptide of the    first aspect, and-   (b) forming an article out of/from the solution provided in (a).

In a sixth aspect, the present invention relates to a pharmaceuticalcomposition comprising the article of the fourth aspect.

In a seventh aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of sixth aspect for useas a pharmaceutical.

In an eight aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of sixth aspect forinducing an immune response.

In a ninth aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of sixth aspect for usein a prophylactic and/or therapeutic treatment of a disease.

In a tenth aspect, the present invention relates to a method fordelivering an antigen to a cell comprising administering to a subjectthe article of the fourth aspect or the pharmaceutical composition ofthe sixth aspect.

In an eleventh aspect, the present invention relates to a method forinducing an immune response in a subject comprising administering to asubject the article of the fourth aspect or the pharmaceuticalcomposition of the sixth aspect.

In a twelfth aspect, the present invention relates to a method forprophylactic and/or therapeutic treatment of a disease in a subjectcomprising administering to a subject the article of the fourth aspector the pharmaceutical composition of the sixth aspect.

In a thirteenth aspect, the present invention relates to a method forstimulating, priming, and/or expanding T cells in a subject comprisingadministering to a subject the article of the fourth aspect or thepharmaceutical composition of the sixth aspect.

This summary of the invention does not necessarily describe all featuresof the present invention. Other embodiments will become apparent from areview of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, GenBank Accession Number sequence submissions etc.),whether supra or infra, is hereby incorporated by reference in itsentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention. In the event of a conflict between the definitions orteachings of such incorporated references and definitions or teachingsrecited in the present specification, the text of the presentspecification takes precedence.

The term “comprise” or variations such as “comprises” or “comprising”according to the present invention means the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers. The term “consisting essentially of” according tothe present invention means the inclusion of a stated integer or groupof integers, while excluding modifications or other integers which wouldmaterially affect or alter the stated integer. The term “consisting of”or variations such as “consists of” according to the present inventionmeans the inclusion of a stated integer or group of integers and theexclusion of any other integer or group of integers.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.

The terms “polypeptide” and “protein” are used interchangeably in thecontext of the present invention. They refer to a long peptide-linkedchain of amino acids, e.g. one that is typically 40 amino acids long orlonger than 40 amino acids.

The term “peptide”, as used herein, refers to a short peptide-linkedchain of amino acids, e.g. one that is typically less than about 40amino acids long and more typically less than about 30 amino acids long.

The terms “fusion protein” or “hybrid protein” (literally, made of partsfrom different sources) are used interchangeably in the context of thepresent invention. They refer to a protein which is created through thejoining of two or more genes that originally coded for separateproteins/peptides. Translation of this fusion gene results in a singlechain polypeptide with functional properties derived from each of theoriginal proteins/peptides.

The terms “recombinant fusion protein” or “recombinant hybrid protein”are used interchangeably in the context of the present invention. Theyrefer to a single chain polypeptide which is created artificially byrecombinant DNA technology.

In one embodiment, the polypeptide of the present invention is a fusionor hybrid polypeptide comprising a silk polypeptide and an antigen. Inone preferred embodiment, the polypeptide of the present invention is afusion or hybrid polypeptide comprising a silk polypeptide, an antigen,and an enzymatically cleavable linker, wherein the antigen is attachedto the silk polypeptide via the enzymatically cleavable linker orwherein the enzymatically cleavable linker is located between the silkpolypeptide and the antigen. The sequence of the enzymatically cleavablelinker may be modified by adding further non-functional amino acids.

The term “silk polypeptide”, as used herein, refers to a polypeptidewhich shows, in comparison to other polypeptides, a quite aberrant aminoacid composition. In particular, a silk polypeptide possess largequantities of hydrophobic amino acids such as glycine or alanine, but,for example, no (or only very little) tryptophan. In addition, a silkpolypeptide contains highly repetitive amino acid sequences orrepetitive units (repeat units, modules), especially in their large coredomain.

Based on DNA analysis, it was shown that all silk polypeptides arechains of repetitive units which further comprise a limited set ofdistinct shorter peptide motifs. The expressions “peptide motif” and“consensus sequence” can be used interchangeably herein. Generally, thesilk consensus sequences can be grouped into four major categories:GPGXX, GGX, A_(x) or (GA)_(n) and spacers. These categories of peptidemotifs in silk polypeptides have been assigned structural roles. Forexample, it has been suggested that the GPGXX motif is involved in aβ-turn spiral, probably providing elasticity. The GGX motif is known tobe responsible for a glycine-rich 3₁-helix. Both GPGXX and GGX motifsare thought to be involved in the formation of an amorphous matrix thatconnects crystalline regions, thereby providing elasticity of the fiber.Alanine-rich motifs typically contain 6-9 residues and have been foundto form crystalline β-sheets. The spacers typically contain chargedgroups and separate the iterated peptide motifs into clusters.

Preferably, the silk polypeptide is a spider silk polypeptide. Morepreferably, the silk polypeptide, e.g. spider silk polypeptide, is arecombinant polypeptide.

The term “antigen”, as used herein, relates to an molecule comprising anepitope against which an immune response is to be generated. The term“antigen” includes proteins or peptides. The term “antigen” alsoincludes molecules, which become antigenic only through transformation(e.g. intermediately in the molecule or by completion with bodyprotein). An antigen is preferably presentable by cells of the immunesystem such as antigen presenting cells (APCs) like dendritic cells(DCs) or macrophages. In addition, an antigen or a processing productthereof is preferably recognizable by a T or B cell receptor, or by animmunoglobulin molecule such as an antibody. The antigen may be adisease-associated antigen, such as a tumor antigen, a viral antigen, ora bacterial antigen.

In the context of the present invention, the term “disease-associatedantigen” is used in its broadest sense to refer to any antigenassociated with a disease. A disease-associated antigen is a moleculewhich contains epitopes that will stimulate a host's immune system tomake a cellular antigen-specific immune response and/or a humoralantibody response against the disease. The disease-associated antigenmay, therefore, be used for therapeutic purposes. Disease-associatedantigens are preferably associated with infection by microbes, typicallymicrobial antigens, or associated with cancer, typically tumors.

The term “disease”, as used herein, refers to an abnormal condition thataffects the body of an individual. A disease is often construed as amedical condition associated with specific symptoms and signs. A diseasemay be caused by factors originally from an external source, such asinfectious disease, or it may be caused by internal dysfunctions, suchas autoimmune diseases. In humans, “disease” is often used more broadlyto refer to any condition that causes pain, dysfunction, distress,social problems, or death to the individual afflicted, or similarproblems for those in contact with the individual. In this broadersense, it sometimes includes injuries, disabilities, disorders,syndromes, infections, isolated symptoms, deviant behaviors, andatypical variations of structure and function, while in other contextsand for other purposes these may be considered distinguishablecategories. Diseases usually affect individuals not only physically, butalso emotionally, as contracting and living with many diseases can alterone's perspective on life, and one's personality.

As mentioned above, the disease may be an infectious disease or anautoimmune disease. The disease may also be a cancer disease or simplycancer.

The term “infectious disease”, as used herein, refers to any diseasewhich can be transmitted from individual to individual or from organismto organism, and is caused by a microbial agent (e.g. common cold).Infectious diseases are known in the art and include, for example, aviral disease, a bacterial disease, or a parasitic disease. Saiddiseases are caused by a virus, a bacterium, and a parasite,respectively. In this regard, the infectious disease can be, forexample, hepatitis, sexually transmitted diseases (e.g. chlamydia orgonorrhea), tuberculosis, HIV/acquired immune deficiency syndrome(AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acuterespiratory syndrome (SARS), the bird flu, and influenza.

The term “autoimmune disease”, as used herein, refers to any disease inwhich the body produces an immunogenic (i.e. immune system) response tosome constituent of its own tissue. In other words, the immune systemloses its ability to recognize some tissue or system within the body asself and targets and attacks it as if it were foreign. Autoimmunediseases can be classified into those in which predominantly one organis affected (e.g. hemolytic anemia and anti-immune thyroiditis), andthose in which the autoimmune disease process is diffused through manytissues (e.g. systemic lupus erythematosus). For example, multiplesclerosis is thought to be caused by T cells attacking the sheaths thatsurround the nerve fibers of the brain and spinal cord. This results inloss of coordination, weakness, and blurred vision. Autoimmune diseasesare known in the art and include, for instance, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma,psoriasis, and the like.

The terms “cancer disease” or “cancer”, as used herein, refer to ordescribe the physiological condition in an individual that is typicallycharacterized by unregulated cell growth. Examples of cancers include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particularly, examples of such cancers include bonecancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, colon cancer, breast cancer, prostate cancer,uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, and pituitary adenoma. The term “cancer” accordingto the invention also comprises cancer metastases.

As mentioned above, the antigen may be a tumor antigen, a viral antigen,or a bacterial antigen.

The term “tumor antigen”, as used herein, refers to a constituent ofcancer cells which may be derived from the cytoplasm, the cell surface,and the cell nucleus. In particular, it refers to those antigens whichare produced, preferably in large quantity, intracellularly or assurface antigens on tumor cells. Examples for tumor antigens includeHER2, EGFR, VEGF, CAMPATH1-antigen, CD22, CA-125, HLA-DR,Hodgkin-lymphoma or mucin-1, but are not limited thereto.

The term “viral antigen”, as used herein, refers to any viral componenthaving antigenic properties, i.e. being able to provoke an immuneresponse in an individual. The viral antigen may be a viralribonucleoprotein or an envelope protein.

The term “microbial antigen”, as used herein, refers to any microbialcomponent having antigenic properties, i.e. being able to provoke animmune response in an individual.

The term “bacterial antigen”, as used herein, refers to any bacterialcomponent having antigenic properties, i.e. being able to provoke animmune response in an individual. The bacterial antigen may be derivedfrom the cell wall or cytoplasm membrane of a bacterium.

The term “fungal antigen”, as used herein, refers to any fungalcomponent having antigenic properties, i.e. being able to provoke animmune response in an individual.

The term “zooparasitic antigen”, as used herein, refers to any componentof a parasite of an animal having antigenic properties, i.e. being ableto provoke an immune response in an individual. Said parasite may be aflea, louse, or worm.

The term “immune response”, as used herein, relates to a reaction of theimmune system to immunogenic organisms, such as bacteria, viruses, cellsor substances. The term “immune response” includes the innate immuneresponse and the adaptive immune response. Preferably, the immuneresponse is related to an activation of immune cells, an induction ofcytokine biosynthesis, and/or antibody production. It is preferred thatthe immune response induced by the articles of the present invention (ormore specifically by the antigens being part of it) comprises the stepsof activation of antigen presenting cells (APCs), such as dendriticcells (DCs) and/or macrophages, presentation of the antigen or fragmentthereof by said antigen presenting cells and activation of cytotoxic Tcells due to this presentation.

The term “immune cells”, as used herein, refers to cells of the immunesystem which are involved in defending the body of an individual. Theterm “immune cells” encompasses specific types of immune cells and theirprecursors including leucocytes comprising macrophages, monocytes(precursors of macrophages), granulocytes such as neutrophils,eosinophils and basophils, dendritic cells, mast cells, and lymphocytessuch as B cells, T cells and natural killer (NK) cells. Macrophages,monocytes (precursors of macrophages), neutrophils, dendritic cells(DCs), and mast cells are phagocytic cells.

The term “antigen presenting cell (APC)”, as used herein, is a cell of avariety of cells capable of displaying, acquiring, and/or presenting atleast one antigen or antigenic fragment on (or at) its cell surface.Antigen-presenting cells can be distinguished in professional antigenpresenting cells and non-professional antigen presenting cells.

The term “professional antigen presenting cells”, as used herein,relates to antigen presenting cells which constitutively express theMajor Histocompatibility Complex class II (MHC class II) moleculesrequired for interaction with naive T cells. If a T cell interacts withthe MHC class II molecule complex on the membrane of the antigenpresenting cell, the antigen presenting cell produces a co-stimulatorymolecule inducing activation of the T cell. Professional antigenpresenting cells comprise dendritic cells (DCs) and macrophages.

The term “non-professional antigen presenting cells”, as used herein,relates to antigen presenting cells which do not constitutively expressMHC class II molecules, but upon stimulation by certain cytokines suchas interferon-gamma. Exemplary, non-professional antigen presentingcells include fibroblasts, thymic epithelial cells, thyroid epithelialcells, glial cells, pancreatic beta cells or vascular endothelial cells.

The term “dendritic cell (DC)”, as used herein, refers to anothersubtype of phagocytic cells belonging to the class of antigen presentingcells (APCs). Preferably, dendritic cells are derived from hematopoieticbone marrow progenitor cells. These progenitor cells initially transforminto immature dendritic cells. These immature cells are characterized byhigh phagocytic activity and low T cell activation potential. Immaturedendritic cells constantly sample the surrounding environment forpathogens such as viruses and bacteria. Once they have come into contactwith a presentable antigen, they become activated into mature dendriticcells and begin to migrate to the spleen or to the lymph node. Immaturedendritic cells phagocytose pathogens and degrade their proteins intosmall pieces and upon maturation present those fragments at their cellsurface using MHC molecules. Simultaneously, they upregulatecell-surface receptors that act as co-receptors in T cell activationsuch as CD80, CD86, and CD40 greatly enhancing their ability to activateT cells. They also upregulate CCR7, a chemotactic receptor that inducesthe dendritic cell to travel through the blood stream to the spleen orthrough the lymphatic system to a lymph node. Here they act asantigen-presenting cells and activate helper T cells and killer T cellsas well as B cells by presenting them antigens, alongside non-antigenspecific co-stimulatory signals. Thus, dendritic cells can activelyinduce a T cell- or B cell-related immune response. Preferably, thedendritic cells are splenic dendritic cells.

The term “macrophage”, as used herein, refers to a subgroup ofphagocytic cells produced by the differentiation of monocytes.Macrophages which are activated by inflammation, immune cytokines, ormicrobial products nonspecifically engulf and kill foreign pathogenswithin the macrophage by hydrolytic and oxidative attack resulting indegradation of the pathogen. Peptides from degraded proteins aredisplayed on the macrophage cell surface where they can be recognized byT cells, and they can directly interact with antibodies on the B cellsurface, resulting in T and B cell activation and further stimulation ofthe immune response. Macrophages belong to the class of antigenpresenting cells (APCs). Preferably, the macrophages are splenicmacrophages.

The terms “T cells” or “T lymphocytes”, as used herein, relate to typesof lymphocytes that play a central role in cell-mediated immunity. Tcells or T lymphocytes can be distinguished from other lymphocytes, suchas B cells and natural killer (NK) cells, by the presence of a T cellreceptor (TCR) on the cell surface. They do not have antigen presentingproperties (but rather, requiring B cells or NK cells for itsantigen-presenting property). They are called T cells because theymature in the thymus. T cells are capable of recognizing an antigen whendisplayed on the surface of antigen presenting cells or matrix togetherwith one or more MHC molecules or one or more non-classical MHCmolecules.

The terms “stimulating T cells” or “stimulation of T cells”, as usedherein, refer to the induction or activation of a T cell response by aprimary signal, such as by the interaction with an antigen-MHC class IIcomplex through the T cell antigen receptor. The term also includes theco-stimulation of T cells, such as through cytokines (e.g. CD80 or CD86proteins). A T cell is activated if it has received a primary signalingevent which initiates an immune response by the T cell.

The term “priming T cells”, as used herein, refers to the induction of afirst contact of the T cell with its specific antigen (e.g. by dendriticcells (DCs) presenting the antigen to T cells), which causes thedifferentiation of the T cell into an effector T cell (e.g. a cytotoxicT cell or a T helper cell).

The terms “expanding T cells” or “expansion of T cells”, as used herein,refer to the increase of the number of T cells, preferably T cellsspecifically recognizing an antigen. It is preferred, that the number ofT cells specifically recognizing the antigen comprised in the article ofthe present invention or the procession product of the antigenincreases. The antigen or procession product of the antigen ispreferably presented in the context of MHC molecules, such as on thesurface of antigen presenting cells (APCs) like dendritic cells (DCs) ormacrophages.

The term “immunotherapy”, as used herein, relates to the treatment of adisease or condition by inducing, enhancing, or suppressing an immuneresponse. Immunotherapies designed to elicit or amplify an immuneresponse are classified as activation immunotherapies, whileimmunotherapies that reduce or suppress an immune response areclassified as suppression immunotherapies. The term “immunotherapy”includes antigen immunization or antigen vaccination as well as tumorimmunization or tumor vaccination. The term “immunotherapy” also relatesto the manipulation of immune responses such that inappropriate immuneresponses are modulated into more appropriate ones in the context ofautoimmune diseases such as rheumatic arthritis, allergies, diabetes, ormultiple sclerosis.

The terms “immunization” or “vaccination”, as used herein, describe theprocess of administering an antigen to an individual with the purpose ofinducing an immune response, for example, for therapeutic orprophylactic reasons.

The term “therapeutic treatment”, as used herein, relates to anytreatment which improves the health status and/or prolongs (increases)the lifespan of an individual. Said treatment may eliminate the diseasein an individual, arrest, inhibit, or slow the development of a diseasein an individual, decrease the frequency or severity of symptoms in anindividual, and/or decrease the recurrence in an individual whocurrently has or who previously has had a disease.

The terms “prophylactic treatment” or “preventive treatment”, as usedherein, relate to any treatment that is intended to prevent a diseasefrom occurring in an individual. The terms “prophylactic treatment” or“preventive treatment” are used herein interchangeably.

The terms “protect”, “prevent”, “prophylactic”, “preventive”, or“protective”, as used herein, relate to the prevention and/or treatmentof the occurrence and/or the propagation of a disease, e.g. tumor, in anindividual. For example, a prophylactic administration of animmunotherapy, e.g. by administering the article or the pharmaceuticalcomposition of the present invention, can protect the receivingindividual from the development of a tumor. For example, a therapeuticadministration of an immunotherapy, e.g. by administering the article orthe pharmaceutical composition of the present invention, can stop thedevelopment of a disease, e.g. lead to the inhibition of theprogress/growth of a tumor. This comprises the deceleration of theprogress/growth of the tumor, in particular a disruption of theprogression of the tumor, which preferably leads to elimination of thetumor. A therapeutic administration of an immunotherapy may protect theindividual from the dissemination or metastasis of existing tumors.

The terms “individual” and “subject” are used interchangeably in thecontext of the present invention. The individual or subject may behealthy, afflicted with a disease or disorder (e.g. cancer), orsusceptible to a disease or disorder (e.g. cancer). The individual orsubject may be an animal, e.g. a human. Preferably, the individual orsubject is a human or another mammal (e.g. mouse, rat, rabbit, dog, cat,cattle, swine, sheep, horse or primate). Unless otherwise stated, theterms “individual” and “subject” do not denote a particular age and,thus, encompass adults, elderlies, children, and newborns. The“individual” or “subject” may be a “patient”.

The term “patient”, as used herein, means an individual or subject whichis diseased, i.e. which suffers from a disease or disorder. The patientmay be an animal, e.g. a human. Preferably, the animal is a human oranother mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep,horse or primate).

The article of the present invention may be administered in the form ofany suitable pharmaceutical composition. Said pharmaceutical compositionmay further comprise adjuvants, pharmaceutical acceptable carriers,diluents, and/or excipients. Said pharmaceutical composition is usefulfor treating, preventing, or reducing the severity of a disease ordisorder. It may be administered locally or systemically, preferablysystemically.

The term “systemic administration”, a used herein, refers to theadministration of the article of the present invention such that thearticle becomes widely distributed in the body of an individual insignificant amounts and develops a biological effect (or morespecifically the antigen comprised therein). Typical systemic routes ofadministration include administration by introducing the articledirectly into the vascular system or oral, pulmonary, or intramuscularadministration wherein the article is adsorbed, enters the vascularsystem, and is carried to one or more desired site(s) of action via theblood.

The systemic administration may be by parenteral administration. Theterm “parenteral administration”, as used herein, refers to theadministration of the article of the present invention such that thearticle does not pass the intestine. The term “parenteraladministration” includes intravenous administration, subcutaneousadministration, intradermal administration, or intraarterialadministration, but is not limited thereto.

As mentioned above, the pharmaceutical compositions of the presentinvention may comprise adjuvants. The term “adjuvant”, as used herein,relates to a compound, which, when administered in combination with anantigen or antigen peptide to an individual, prolongs, enhances, oraccelerates an immune response. It is assumed that adjuvants exert theirbiological activity by one or more mechanisms, including an increase ofthe surface of the antigen, a prolongation of the retention of theantigen in the body, a retardation of the antigen release, targeting ofthe antigen to macrophages, increase of the uptake of the antigen,enhancement of antigen processing, stimulation of cytokine release,stimulation and activation of immune cells such as B cells, macrophages,dendritic cells, T cells and unspecific activation of immune cells.Adjuvants comprise a heterogeneous group of compounds such as oilemulsions (e.g., Freund's adjuvants), mineral compounds (such as alum),bacterial products (such as Bordetella pertussis toxin), orimmune-stimulating complexes. Examples for adjuvants include saponins,incomplete Freund's adjuvants, complete Freund's adjuvants, tocopherols,or aluminium, but are not limited thereto. However, adjuvants may alsohave negative side effects. Aluminium salt, for example, has thepotential to cause severe local and systemic side-effects includingsterile abscesses, eosinophilia and myofascitis, although fortunatelymost of the more serious side-effects are relatively rare. In addition,there is also community concern regarding the possible role of aluminiumin neurodegenerative diseases such as Alzheimer's disease. Consequently,there is a major unmet need for safer and more effective adjuvantssuitable for human use. The best would be, if the use of adjuvants wouldbe superfluous.

The pharmaceutical composition according to the present invention isgenerally applied in a “pharmaceutically effective amount”. The term“pharmaceutically effective amount”, as used herein, refers to theamount which achieves a desired reaction or a desired effect alone ortogether with further doses. In case of the treatment of a particulardisease, the desired reaction preferably relates to an inhibition of thecourse of the disease. This comprises slowing down the progress of thedisease and, in particular, interrupting or reversing the progress ofthe disease. The desired reaction in a treatment of a disease may alsobe a delay of the onset or a prevention of the onset of the disease. Aneffective amount of the articles or compositions described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient/subject, including age,physiological condition, size, and weight, the duration of treatment,the type of an accompanying therapy (if present), the specific route ofadministration, and similar factors. Accordingly, the doses of thearticles or compositions described herein may depend on various of suchparameters. In case that a reaction in the patient/subject isinsufficient with an initial dose, higher doses (or effectively higherdoses achieved by a different, more localized route of administration)may be used.

As mentioned above, the pharmaceutical composition of the presentinvention may further comprise pharmaceutical acceptable carriers,diluents, and/or excipients.

The term “excipient”, as used herein, is intended to indicate allsubstances in a pharmaceutical composition which are not activeingredients such as binders, lubricants, thickeners, surface activeagents, preservatives, emulsifiers, buffers, flavoring agents, orcolorants.

The term “diluent”, as used herein, relates to a diluting and/orthinning agent. Moreover, the term “diluent” includes a solution,suspension (e.g. liquid or solid suspension) and/or media.

The term “carrier”, as used herein, relates to one or more compatiblesolid or liquid fillers, which are suitable for an administration, e.g.to a human. The term “carrier” relates to a natural or synthetic organicor inorganic component which is combined with an active component inorder to facilitate the application of the active component. Preferably,carrier components are sterile liquids such as water or oils, includingthose which are derived from mineral oil, animals, or plants, such aspeanut oil, soy bean oil, sesame oil, sunflower oil, etc. Salt solutionsand aqueous dextrose and glycerin solutions may also be used as aqueouscarrier compounds.

Pharmaceutically acceptable carriers or diluents for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaroedit. 1985). Examples of suitable carriers include, for example,magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Examples of suitable diluents include ethanol, glycerol, and water.

Pharmaceutical carriers, diluents, and/or excipients can be selectedwith regard to the intended route of administration and standardpharmaceutical practice. The pharmaceutical compositions of the presentinvention may comprise as, or in addition to, the carrier(s),excipient(s) or diluent(s) any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), and/or solubilising agent(s).Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose, and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,and the like. Preservatives, stabilizers, dyes, and even flavoringagents may be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid, and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused.

In the context of the present invention, the term “particle” refers to astructured entity formed by polypeptides. According to the presentinvention, the structured entity formed by polypeptides comprises silkpolypeptides and antigens. In one embodiment, the particle comprises afusion or hybrid polypeptide comprising a silk polypeptide and anantigen. In one preferred embodiment, the particle comprises a fusion orhybrid polypeptide comprising a silk polypeptide, an antigen, and anenzymatically cleavable linker, wherein the antigen is connected to thesilk polypeptide via the enzymatically cleavable linker or wherein theenzymatically cleavable linker is located between the silk polypeptideand the antigen.

The term “particle”, as used herein, further refers to a micro- ornano-sized spherical structure which may be formed by proteinaggregation under certain conditions. It is preferred that the particlecomprises or consists of a matrix and a surface. Preferably, the matrixis homogenous, more preferably without any clear visible inclusions(e.g. determined via electron microscopy). In this respect, it should benoted that said inclusions may be air and polypeptides which are notrelated to the polypeptides of the present invention.

The term “surface”, as used herein, defines the outer sphere of theparticle, which includes those sphere sections that are directly exposedto the surrounding space, e.g. surrounding medium or body liquid.Although the particle appears rather smooth and uniform, its surface onthe sub-microscopic level reveal a thin mantle with irregular anddiffuse structures. A surface, thus, delineates the outermost layer ofthe particle which shares an interface with the surrounding space, e.g.surrounding medium or body liquid.

The term “matrix”, as used herein, defines the inner sphere of theparticle, which is not the surface, i.e. which, according to the abovedefinition, does not include any interface to the surrounding space,e.g. surrounding medium or body liquid. The matrix is to be understoodas a solid sphere having a radius and accordingly a volume usuallysmaller than that of the particle. The volume of the matrix is usuallymore than 50% of the total volume, preferably more than 60%, 70%, 80%,90%, most preferably more than 95%, e.g. more than 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, or 95%.

The terms “aggregation” or “phase separation”, as used herein, refer toparticle formation due to a salting-out mechanism which in particularcan be considered as a liquid-liquid phase separation. The “one-phasestate” is the initial state displayed by a solution of monomeric andintrinsically unfolded protein molecules. For example, changingconstraints such as the ionic strength by addition of kosmotropic ionsalters the free energy of the system and leads to phase separation intoprotein-rich and solvent-rich phases. This phase-separated state isenergetically favoured and the protein concentration in the “proteinphase” increases to a critical level. Upon reaching the criticalconcentration for nucleation, several structured nuclei are formedsimultaneously in the protein-rich phase. The nuclei start to grow in aspherical manner, interacting with additional monomers and therebyconverting their structure. Spherical growth stops when the proteinconcentration in the protein-rich phase is below the equilibrium ofsolubility. Hence the sphere size does not increase further. Phaseseparation, thus, means that protein-rich and solvent-rich phases areseparated. Without being bound to a theory, the sphere size is generallydependent on protein concentration and mixing conditions. There existhowever various other methods in the art for triggering aggregation ofproteins.

The process of microsphere assembly is typically monitored bylight-scattering after initiation of aggregation. In particular, thecolloidal stability of the resulting particles can be analysed bymeasuring the intensity of scattered light at a certain wavelength. Alsothe mean particle size and particle size distribution can be determinedby laser diffraction, also called static light scattering (SLS).

The obtained particles may also be analysed using methods such asscanning electron microscopy (SEM) and Fourier transform infraredspectroscopy (FTIR). A further description of these methods can be foundin the description and in the examples below.

After phase separation, the produced particles can be separated byroutine methods such as centrifugation, filtration, or sedimentation.The prepared particles may subsequently be washed and/or stored, forexample, in a dried or lyophilized form.

The term “net charge of the surface of the particle” relates to thetotal sum of charges, such as positive and negative charges, at thesurface of the particle. For example, if the particle comprises on itssurface a higher number of negative charges than positive charges, thenet charge of the surface of the particle is negative. If the particlecomprises on its surface a higher number of positive charges thannegative charges, the net charge of the surface of the particle ispositive. If the particle comprises on its surface an equal number ofpositive charges and negative charges, the net charge of the surface ofthe particle is neutral, particularly electroneutral. Thus, the netcharge of the surface of the particle according to the present inventioncan be negative, positive, or neutral. Preferably, the particle of thepresent invention has a net negative surface charge.

The term “average diameter” refers to the mean diameter of the particlesand may be calculated by dividing the sum of the diameter of eachparticle by the total number of particles. Although the term “diameter”is used normally to refer to the maximal length of a line segmentpassing through the center and connecting two points on the periphery ofa spherical object, it is also used herein to refer to the maximallength of a line segment passing through the center and connecting twopoints on the periphery of particles having a substantial sphericalshape or other shapes.

The term “protease (also designated as peptidase or proteinase)”, asused herein, refers to any enzyme that performs proteolysis, that is,begins protein catabolism by hydrolysis of the peptide bonds that linkamino acids together in a polypeptide chain. Proteases have evolvedmultiple times, and different classes of protease can perform the samereaction by completely different catalytic mechanisms. Proteases can befound in animals, plants, bacteria, archaea and viruses.

The term “cathepsin (CTS)”, as used herein, refers to a protease(enzymes that degrade proteins) which is found in all animals as well asother organisms. Different family members exists, which aredistinguished by their structure, catalytic mechanism, and whichproteins they cleave. Most of the members become activated at the low pHfound in lysosomes. Thus, the activity of this family lies almostentirely within those organelles. Cathepsin may be cathepsin A,cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F,cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2,cathepsin 0, cathepsin S, cathepsin W, or cathepsin Z. Preferably, thecathepsin is cathepsin S or B. More preferably, cathepsin is cathepsinS.

In one embodiment, the polypeptide of the present invention or thepolypeptide used in the present invention comprises a protease cleavablelinker, e.g. a cathepsin S cleavable linker or cathepsin B cleavablelinker. Preferably, the cathepsin S cleavable linker has the sequenceaccording to SEQ ID NO: 1 or is a variant thereof or has the sequenceaccording to SEQ ID NO: 2 or is a variant thereof, or the cathepsin Bcleavable linker has the sequence according to SEQ ID NO: 2 or is avariant thereof. In this respect, it should be noted that cathepsin S isable to cleave its own cleavage sites (comprised in SEQ ID NO: 1) andalso the cleavage sites which are usually cleaved by cathepsin B(comprised in SEQ ID NO: 2). The sequence according to SEQ ID NO: 1(GPMGLPG) comprises the cleavage site PMGLP (SEQ ID NO: 20) and thesequence according to SEQ ID NO: 2 (GAVGFLGIG) comprises the cleavagesite GFLG (SEQ ID NO: 21). Instead of the cleavage site GFLG (SEQ ID NO:21), the dipeptide sequence Val-Cit, or the tetra peptides ALAL (SEQ IDNO: 22) or GGGF (SEQ ID NO: 23) can be used as cathepsin B cleavagesites. The cleavage sites Val-Cit, ALAL (SEQ ID NO: 22), or GGGF (SEQ IDNO: 23) may be effective in combination with the C16 CathBseq-SIINFEKL(SEQ ID NO: 15) particles. In this case, the cleavage site GFLG (SEQ IDNO: 21) within the sequence according to SEQ ID NO: 15 is replaced byVal-Cit, ALAL (SEQ ID NO: 22), or GGGF (SEQ ID NO: 23). It is alsopossible that the cathepsin cleavable linker comprised in thepolypeptide of the present invention consists of the cleavage siteVal-Cit or the cleavages sites according to SEQ ID NO: 20 to 23.

Embodiments of the Invention

The inventors of the present patent application surprisingly found thatantigens which are part of articles, e.g. particles, comprising silkpolypeptides are preferentially taken up by animal cells, e.g. humancells. In this context, the inventors of the present patent applicationwere surprised that the articles, e.g. particles, which are mainlycomposed of silk, function as an auxiliary material which stabilizes theantigen and makes it transportable. The articles, e.g. particles, can bedegraded within the organism (e.g. human body) without traces. Thearticles, e.g. particles, are non-toxic, in particular non-cytotoxic,and non-immunogenic.

Thus, in a first aspect, the present invention relates to a polypeptidecomprising

-   (i) a silk polypeptide and-   (ii) an (polypeptide) antigen.

The polypeptide may be a molecule which is composed of at least twocomponents which can either be translated as a single chain polypeptidefrom the same mRNA molecule or can be produced by separate translationof the at least two components and subsequent coupling, e.g. by chemicalreactions. In the first case, the silk polypeptide is fused/attached tothe antigen. In the second case, the silk polypeptide is linked/coupledto the antigen. Preferably, the polypeptide is a single chainpolypeptide which may also be designated as a hybrid polypeptide orfusion polypeptide.

In one preferred embodiment, the polypeptide further comprises anenzymatically cleavable linker. Said linker may be any linker which iscleavable by an enzyme. When the enzymatically cleavable linker ispresent, the antigen is connected to the silk polypeptide via saidlinker or said linker is located between the silk polypeptide and theantigen. If the polypeptide is produced as a single chain polypeptide(which may also be designated as a hybrid polypeptide or fusionpolypeptide), the antigen is attached/fused to the silk polypeptide viathe enzymatically cleavable linker. If the polypeptide is produced byseparate translation of at least two components and subsequent coupling,e.g. by chemical reactions, the antigen is linked/coupled to the silkpolypeptide via the enzymatically cleavable linker. It is particularlypreferred that the enzymatically cleavable linker is intracellularlycleavable by an enzyme, e.g. within the host cell.

Preferably, the enzymatically cleavable linker is a protease cleavablelinker. Said linker may be any linker which is cleavable by a protease.

More preferably, the protease cleavable linker is a cathepsin cleavablelinker. The cathepsin may be cathepsin A, cathepsin B, cathepsin C,cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H,cathepsin K, cathepsin L1, cathepsin L2, cathepsin 0, cathepsin S,cathepsin W, or cathepsin Z. The cathepsin cleavable linker may be acathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E,cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1,cathepsin L2, cathepsin 0, cathepsin S, cathepsin W, or cathepsin Zcleavable linker.

Even more preferably, the cathepsin cleavable linker is a cathepsin Bcleavable linker or a cathepsin S cleavable linker. The cathepsin Scleavable linker is particularly preferred.

Cathepsin B is a member of the peptidase Cl family. It is a lysosomalcysteine protease with both endopeptidase and exopeptidase activity. Itplays a role in protein turnover. It is also known as amyloid precursorprotein secretase and is involved in the proteolytic processing ofamyloid precursor protein (APP). Cathepsin B is ubiquitously expressedin almost every tissue. In contrast thereto, cathepsin S is onlyexpressed in certain tissues. In particular, cathepsin S is expressed byantigen presenting cells including macrophages, B-lymphocytes, dendriticcells, and microglia. In addition, cathepsin S is expressed by someepithelial cells. Cathepsin S is also a member of the peptidase Clfamily. It is a lysosomal cysteine protease. It plays a key role in thedegradation of antigenic proteins and their further processing via theMHC class II pathway. Cathepsin S is able to cleave its own cleavagesites (see, for example, SEQ ID NO: 1) and also the cleavage sites whichare usually cleaved by cathepsin B (see, for example, SEQ ID NO: 2). Inthis respect, it should be noted that cathepsin S cleaves the cleavagesites which are usually cleaved by cathepsin B slower and in a lessextent.

Most preferably,

-   (i) the cathepsin S cleavable linker has the sequence according to    SEQ ID NO: 1 or is a variant thereof or has the sequence according    to SEQ ID NO: 2 or is a variant thereof, or-   (ii) the cathepsin B cleavable linker has the sequence according to    SEQ ID NO: 2 or is a variant thereof.

The inventors of the present patent application found that cathepsin Sis able to cleave a cathepsin S cleavable linker, e.g. a cathepsin Scleavable linker having the sequence according to SEQ ID NO: 1, as wellas a cathepsin B cleavable linker, e.g. a cathepsin B cleavable linkerhaving the sequence according to SEQ ID NO: 2.

A SEQ ID NO: 1 or SEQ ID NO: 2 variant differs from the referencesequence from which it is derived by up to 1, 2, 3, or 4 amino acidchanges in the amino acid sequence (i.e. substitutions, additions,insertions, deletions, N-terminal truncations and/or C-terminaltruncations). Such a variant can alternatively or additionally becharacterised by a certain degree of sequence identity to the referencemodule from which it is derived. Thus, a SEQ ID NO: 1 or SEQ ID NO: 2variant has a sequence identity of at least 60%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 99.9%to the respective reference sequence. Preferably, the sequence identityis over the whole length of the respective sequence.

A fragment (or deletion variant) of SEQ ID NO: 1 or SEQ ID NO: 2 haspreferably a deletion of up to 1, 2, 3, or 4 amino acids at itsN-terminus and/or at its C-terminus. The deletion can also beinternally.

Additionally, the SEQ ID NO: 1 or SEQ ID NO: 2 variant or fragment isonly regarded as a SEQ ID NO: 1 or SEQ ID NO: 2 variant or fragmentwithin the context of the present invention, if the modifications withrespect to the amino acid sequence on which the variant or fragment isbased do not negatively affect the ability of cathepsin, e.g. cathepsinS or B, to cleave this sequence. The skilled person can readily assesswhether cathepsin S or B is still capable of cleaving the SEQ ID NO: 1or SEQ ID NO: 2 variant, e.g. by performing an enzyme assay on a testsubstrate comprising the modified sequence.

The silk polypeptide may be a spider silk polypeptide, e.g. a majorampullate silk polypeptide such as a dragline silk polypeptide, a minorampullate silk polypeptide, or a flagelliform silk polypeptide of anorb-web spider (e.g. Araneidae or Araneoids), an insect silkpolypeptide, a mussel byssus silk polypeptide, or a mixture thereof. Theorb-web spider may be selected from the group consisting of Araneusdiadematus, Nephila clavipes, and Latrodectus hesperus. The insect silkpolypeptide may be of Lepidoptera, particularly Bombycidae such asBombyx mori. The insect silk polypeptide may also be of Hymenoptera,particularly Apoidea such as Anthophila. Preferably, the silkpolypeptide is a spider silk polypeptide.

It is preferred that the silk polypeptide is a polypeptide with an aminoacid sequence which comprises or consists of at least 50%, 60%, 65%,70%, 75%, 80%, 85%, or 90% multiple copies of repetitive units. It ismore preferred that the silk polypeptide is a polypeptide with an aminoacid sequence which comprises or consists of at least 95% multiplecopies of repetitive units. Said repetitive units may be identical ordifferent. It is particularly preferred that the silk polypeptidecomprises at least two identical repetitive units. For example, the silkpolypeptide may comprise between 2 to 100 repetitive units, e.g. 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100 repetitive units.

It is further (alternatively or additionally) preferred that the silkpolypeptide consists of between 40 to 3000 amino acids. It is morepreferred that the silk polypeptide consists of between 40 to 1500 aminoacids. It is even more preferred that the silk polypeptide consists ofbetween 200 to 1200 amino acids. It is most preferred that the silkpolypeptide consists of between 250 to 600 amino acids.

It is also (alternatively or additionally) preferred that the silkpolypeptide is a recombinant silk polypeptide.

As mentioned above, it is particularly preferred that the silkpolypeptide comprises at least two identical repetitive units. In oneembodiment, the repetitive units are independently selected from thegroup consisting of module C (SEQ ID NO: 3) or a variant thereof, moduleC^(Cys) (SEQ ID NO: 4), and module C^(kappa) (SEQ ID NO: 18). ModuleC^(Cys) (SEQ ID NO: 4) is a variant of module C (SEQ ID NO: 3). In thismodule, the amino acid S (Ser) at position 25 has been replaced by theamino acid C (Cys). Module C^(kappa) (SEQ ID NO: 18) is also a variantof module C (SEQ ID NO: 3). In this module, the amino acid E (Glu) atposition 20 has been replaced by the amino acid K (Lys).

The module C variant differs from the reference module C from which itis derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15amino acid changes in the amino acid sequence (i.e. substitutions,additions, insertions, deletions, N-terminal truncations and/orC-terminal truncations). Such a module variant can alternatively oradditionally be characterised by a certain degree of sequence identityto the reference module from which it is derived. Thus, the module Cvariant has a sequence identity of at least 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 99.9% to the respective reference module C.Preferably, the sequence identity is over a continuous stretch of atleast 5, 10, 15, 18, 20, 24, 27, 28, 30, 34, 35, or more amino acids,preferably over the whole length of the respective reference module C.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference module C. It is further particularlypreferred that the sequence identity is at least 80% over a continuousstretch of at least 5, 10, 15, 18, 20, 24, 28, or 30 amino acids, is atleast 85% over a continuous stretch of at least 5, 10, 15, 18, 20, 24,28, or 30 amino acids, is at least 90% over a continuous stretch of atleast 5, 10, 15, 18, 20, 24, 28, or 30 amino acids, is at least 95% overa continuous stretch of at least 5, 10, 15, 18, 20, 24, 28, or 30 aminoacids, is at least 98% over a continuous stretch of at least 5, 10, 15,18, 20, 24, 28, or 30 amino acids, or is at least 99% over a continuousstretch of at least 5, 10, 15, 18, 20, 24, 28, or 30 amino acids of therespective reference module C.

A fragment (or deletion) variant of module C has preferably a deletionof up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 aminoacids at its N-terminus and/or at its C-terminus. The deletion can alsobe internally.

Additionally, the module C variant or fragment is only regarded as amodule C variant or fragment within the context of the presentinvention, if the modifications with respect to the amino acid sequenceon which the variant or fragment is based do not negatively affect theability of the silk polypeptide to internalize the antigen into thetarget cell. The skilled person can readily assess whether the silkpolypeptide comprising a module C variant or fragment is still capableof internalizing the antigen into the target cell, e.g. by culturingtarget cells, e.g. BMDCs, with fluorescently-labelled, e.g.FITC-labelled, spider silk particles comprising the antigen andconducting flow cytometry analysis (see example 11 of the experimentalsection).

C^(Cys) or C^(kappa) variants may also be encompassed by the presentinvention. Regarding the C^(Cys) or C^(kappa) variants, the sameexplanations/definitions apply which have been made with respect to themodule C variant (see above).

The use of a positively charged protein such as C^(kappa) (SEQ ID NO:18) in contrast to the use of module C (SEQ ID NO: 3) or a variantthereof results in a positive surface charge of the articles at neutralpH. The positive surface charge can improve the uptake into cells.

It is also particularly preferred that the silk polypeptide comprises atleast one non-repetitive (NR) unit. Said non-repetitive (NR) unit may becomprised at the N- and/or C-terminus. In one embodiment, the NR unit isselected from the group consisting of NR3 (SEQ ID NO: 7) or a variantthereof, NR4 (SEQ ID NO: 8) or a variant thereof, NR5 (SEQ ID NO: 9) ora variant thereof, and NR6 (SEQ ID NO: 10) or a variant thereof. The NR3(SEQ ID NO: 7) unit is based on the amino acid sequence of ADF-3 of thespider Araneus diadematus and the NR4 (SEQ ID NO: 8) unit is based onthe amino acid sequence of ADF-4 of the spider Araneus diadematus (WO2006/008163). In addition, the NR5 (SEQ ID NO: 9) unit and the NR6 (SEQID NO: 10) unit are derived from Latrodectus hesperus.

Regarding the NR3, NR4, NR5, or NR6 unit variant, the sameexplanations/definitions apply which have been made with respect to themodule C variant (see above).

In addition, a NR3, NR4, NR5, or NR6 unit variant or fragment is onlyregarded as a NR3, NR4, NR5, or NR6 unit variant or fragment within thecontext of the present invention, if the modifications with respect tothe amino acid sequence on which the variant or fragment is based do notnegatively affect the ability of the silk polypeptide to internalize theantigen into the target cell. The skilled person can readily assesswhether the silk polypeptide comprising a NR3, NR4, NR5, or NR6 unitvariant or fragment is still capable of internalizing the antigen intothe target cell, e.g. by culturing target cells, e.g. BMDCs, withfluorescently-labelled, e.g. FITC-labelled, spider silk particlescomprising the antigen and conducting flow cytometry analysis (seeexample 11 of the experimental section).

It is further particularly preferred that the silk polypeptide comprisesat least one Tag (sequence). The Tag may be comprised at the N- and/orC-terminus. The Tag is preferably non-repetitive. It is suitable for thepurification and/or detection of the silk polypeptide. The Tag may be aHis-Tag or a T7-Tag. Preferably, the T7-Tag has a sequence according toSEQ ID NO: 5 or a sequence according to SEQ ID NO: 6.

In one preferred embodiment, the silk polypeptide is selected from thegroup consisting of (C)_(m), (C^(Cys))_(m), (C^(kappa))_(m),(C)_(m)C^(Cys), C^(Cys)(C)_(m), (C)_(m)C^(Cys)(C)_(m), (C)_(m)NR_(z),NR_(z)(C)_(m) and NR_(z)(C)_(m)NR_(z), wherein m is an integer of 8 to96, i.e. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95or 96, z is an integer of 1 to 3, i.e. 1, 2, or 3, and NR stands for anon-repetitive unit.

In one more preferred embodiment, the silk polypeptide is selected fromthe group consisting of C₈, C₁₆, C₃₂, C₄₈, C^(kappa) ₈, C^(kappa) ₁₆,C^(kappa) ₃₂, C^(kappa) ₄₈, C₈C^(Cys), C₁₆C^(Cys), C₃₂C^(Cys), andC₄₈C^(Cys).

Preferably, the antigen is a disease-associated antigen. Morepreferably, the antigen, in particular the disease-associated antigen,is selected from the group consisting of a viral antigen, a microbialantigen, such as a bacterial or fungal antigen, a zooparasitic antigen,and a tumor antigen.

As an exemplarily antigen, the inventors of the present patentapplication used an epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄) with theamino acid sequence SIINFEKL (SEQ ID NO: 11). The chicken-Ovalbuminepitope with the amino acid sequence SIINFEKL (SEQ ID NO: 11) wasselected, because it is the best characterized and generally appliedsystem to demonstrate/monitor the effect of peptide-epitopes to animmune reaction. This chicken-Ovalbumin epitope system with the aminoacid sequence SIINFEKL (SEQ ID NO: 11) is a state of the art-tool andresembles a universally valid example for the presentation of a plethoraof antigens to the immune system. For the experiments shown herein, theinventors of the present patent application used a recombinant spidersilk polypeptide comprising C₁₆ having the sequence according to SEQ IDNO: 12. The inventors of the present patent application recombinantlyproduced different polypeptides comprising a silk polypeptide and anantigen or a silk polypeptide, an enzymatically cleavable linker, and anantigen. The polypeptide comprising the spider silk polypeptide C₁₆ andan epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄) has the sequence accordingto SEQ ID NO: 13. The polypeptide comprising the spider silk polypeptideC₁₆, a cathepsin S cleavable linker, and an epitope of chicken-Ovalbumin(OVA₂₅₇₋₂₆₄) has the sequence according to SEQ ID NO: 14. Thepolypeptide comprising the spider silk polypeptide C₁₆, a cathepsin Bcleavable linker, and an epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄) hasthe sequence according to SEQ ID NO: 15.

In a second aspect, the present invention relates to a nucleic acidmolecule encoding the polypeptide of the first aspect.

It is preferred that the nucleic acid molecule is comprised in a vector.The vector may be any vector known to the skilled person. For example,the vector may be a plasmid vector, a viral vector such as an adenoviralor a baculoviral vector, a cosmid vector, or a phage vector such as alambda phage vector. Said vectors include expression as well as cloningvectors. Expression vectors generally contain a desired coding sequenceand appropriate DNA sequences to control the expression of the operablylinked coding sequence in a particular host organism (e.g., bacteria,yeast, plant, insect, or mammal) or in in vitro expression systems.Expression control sequences may be sequences which control (i) theexpression, e.g. promoters, TATA-box, enhancers, (ii)post-transcriptional events, e.g. polyadenylation, and (iii) thetranslation of nucleic acid sequences. Cloning vectors are generallyused to engineer and amplify a certain desired DNA fragment and may lackfunctional sequences needed for expression of the desired DNA fragments.The above-mentioned vectors are preferably recombinant vectors.

The cell may be transformed, transfected, or infected with the nucleicacid molecule or the vector comprising the nucleic acid molecule. Thecell can be used for expressing the nucleic acid molecule or amplifyingthe nucleic acid molecule or the vector comprising the nucleic acidmolecule. The cell may be a prokaryotic or eukaryotic cell. Theprokaryotic cell may be a E. coli cell or a Bacillus subtilis cell. Theeukaryotic cell may be a mammalian cell, a plant cell, a yeast cell, oran insect cell. The mammalian cell may be a CHO, COS, HeLa, 293T, HEH,or BHK cell. The yeast cell may be a Saccharomyces cerevisiae cell, aSchizosaccharomyces pombe cell, a Pichia pastoris cell, a Candidaalbicans cell, or a Hansenula polymorpha cell. The insect cell may be aLepidoptera insect cell. The plant cell may be a tobacco cell, a potatocell, a corn cell, a pea cell, or a tomato cell. The above-mentionedcells may also be named host cells. They are preferably recombinantcells.

In a third aspect, the present invention relates to a method forproducing a polypeptide comprising the step of:

-   (a) expressing the nucleic acid molecule of the second aspect in a    cell, thereby producing the polypeptide in the cell.

The cell may also be named host cell. The cell may be a prokaryotic oreukaryotic cell. The prokaryotic cell may be a E. coli cell or aBacillus subtilis cell. The eukaryotic cell may be a mammalian cell, aplant cell, a yeast cell, or an insect cell. The mammalian cell may be aCHO, COS, HeLa, 293T, HEH, or BHK cell. The yeast cell may be aSaccharomyces cerevisiae cell, a Schizosaccharomyces pombe cell, aPichia pastoris cell, a Candida albicans cell, or a Hansenula polymorphacell. The insect cell may be a Lepidoptera insect cell. The plant cellmay be a tobacco cell, a potato cell, a corn cell, a pea cell, or atomato cell. The above-mentioned cells are preferably recombinant cells.

The nucleic acid molecule may be found inside the cell (i) freelydispersed as such, (ii) incorporated in a vector, or (iii) integratedinto the cell genome or mitochondrial DNA. Preferably, the vector, e.g.plasmid or viral vector, is an expression vector.

The cell may be transformed, transfected, or infected with the nucleicacid molecule or the vector comprising the nucleic acid molecule.

Preferably, the method further comprises the step of:

-   (b) isolating the polypeptide from the cell.

The isolation of the polypeptide from the cell may be achieved byseparating said polypeptide from said cell via centrifugation,sedimentation, and/or filtration, e.g. via centrifugation andfiltration, via sedimentation and filtration, via sedimentation andcentrifugation, or via centrifugation, sedimentation, and filtration.Depending on the polypeptide to be harvested, the parameters forcentrifugation, sedimentation, or filtration may vary. The personskilled in the art is able to easily adapt the appropriate separationparameters, e.g. the acceleration-force/G-force and/or time usingcentrifugation for separation, filter size using filtration forseparation, and/or sedimentation time using sedimentation forseparation, in order to harvest said polypeptide produced by said cells.

After step (b), further purification of the isolated polypeptide may berequired. Said purification may be achieved via chromatography,preferably column chromatography, more preferably size exclusionchromatography, hydrophobic interaction chromatography, ion exchangechromatography, affinity chromatography, or high pressure liquidchromatography, electrophoresis, preferably gel electrophoresis, orultracentrifugation. A preferred isolation method for the production ofpolypeptides is described in WO 2011/120690 A2.

In one preferred embodiment, the purified polypeptide is endotoxin free.Endotoxin depletion is achieved by filtration, steam sterilisation (e.g.in an autoclave), or a combination of both, i.e. filtration and steamsterilisation (e.g. in an autoclave).

In a fourth aspect, the present invention relates to an articlecomprising the polypeptide of the first aspect. The article may also benamed antigen carrier or antigen carrier vehicle.

The article may be selected from the group consisting of a particle,capsule, fiber, film, granule, gel, fabric made of fibers, rod orbundles thereof. Preferably, the rod comprises or consists of fibers.Preferably, the fabric is a woven or non-woven fabric. It is preferredthat said article is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that said article issterilizable.

Preferably, said article is a particle. It is preferred that theparticle is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that the particle issterilizable. The particle is preferably a nanoparticle, becausecellular uptake is a major aspect of its action path and, therefore,desirable.

In one embodiment, the particle has an average diameter in the range offrom 50 nm to 1000 nm, e.g. from 100 nm to 900 nm, from 200 nm to 800nm, from 200 to 700 nm, from 300 to 600 nm, from 300 nm to 500 nm, orfrom 300 nm to 400 nm. The range of from 250 to 520 nm is particularlypreferred.

In one further embodiment, the particle has an average diameter of atleast 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm,at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, atleast 700 nm, at least 800 nm, at least 900 nm, and/or the particle hasan average diameter of no more than 1000 nm, no more than 900 nm, nomore than 800 nm, no more than 700 nm, no more than 600 nm, no more than520 nm, no more than 500 nm, no more than 400 nm, no more than 300 nm,no more than 250 nm, no more than 200 nm, no more than 150 nm, no morethan 100 nm, no more than 90 nm, no more than 80 nm, no more than 70 nm,no more than 60 nm.

In one preferred embodiment, the particle has an average diameter (i) inthe range of from 50 nm to 400 nm, preferably from 50 nm to 200 nm, or(ii) in the range of from 200 nm to 1000 nm, preferably from 200 nm to800 nm, more preferably from 250 nm to 520 nm or from 300 nm to 600 nm.

In a fifth aspect, the present invention relates to a method forproducing an article comprising the steps of:

-   (a) providing an aqueous solution comprising the polypeptide of the    first aspect, and-   (b) forming an article out of/from the solution provided in (a).

Said article may also be named antigen carrier or antigen carriervehicle.

As the silk polypeptide and the antigen are part of the samepolypeptide, no additional loading step is necessary after articleformation.

The articles, which are mainly composed of silk, function as anauxiliary material which stabilizes the antigen and makes ittransportable. The articles can be degraded within the organism (e.g.human body) without traces.

The above-described article formation process does not require organicsolvents. The avoidance of organic solvents has the advantage thatorganic solvent sensitive antigens retain their characteristicproperties.

Preferably, the concentration of the polypeptide in the aqueous solutionis between 0.1 wt %/vol and 30 wt %/vol, more preferably between 1 wt%/vol and 20 wt %/vol, even more preferably between 1 wt %/vol and 20 wt%/vol, and most preferably between 2 to 8 wt %/vol or 4 to 6 wt %/vol.Thus, for example, the concentration of the polypeptide in the aqueoussolution is 0.1, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4,2.6, 2.8, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0,10.0, 12.0, 15.0, 18.0, 20.0, 25.0, or 30.0 wt %/vol.

The aqueous solution may be a buffered aqueous solution or water (H₂O)such as technical H₂O or deionized H₂O. The buffered aqueous solutionmay be, for example, Tris/HCl. Preferably, the pH of the bufferedaqueous solution is between pH 5.0 and pH 9.0, more preferably betweenpH 6.0 and pH 8.0, and even more preferably between pH 6.7 and pH 7.2,e.g. Tris/HCl, pH 7.0, pH 7.5, or pH 8.0. Preferably, the bufferedaqueous solution is a solution between 10 and 100 mM Tris/HCl, morepreferably between 10 and 50 mM Tris/HCl, and most preferably between 10and 20 mM Tris/HCl, e.g. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 mM Tris/HCl.

Said article may be selected from the group consisting of a particle,capsule, fiber, film, granule, gel, fabric made of fibers, rod orbundles thereof. Preferably, the rod comprises or consists of fibers.Preferably, the fabric is a woven or non-woven fabric. It is preferredthat said article is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that said article issterilizable.

In one embodiment, the article is a particle. It is preferred that theparticle is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that the particle issterilizable. Generally, starting from an aqueous solution, aggregationof the polypeptide can be triggered under certain conditions to formparticles. When the article is a particle, step (b) comprises triggeringaggregation of the polypeptide. Thus, in one preferred embodiment, themethod is for producing a particle and comprises the steps of:

-   (a) providing an aqueous solution comprising the polypeptide of the    first aspect, and-   (b) forming a particle out of/from the solution provided in (a),    wherein said formation comprises triggering aggregation of the    polypeptide.

The aggregation may be triggered by pH shift, ion exchange, shearforces, the addition of alcohol, the addition of a salt, the removal ofwater, temperature change, and by combinations thereof.

The alcohol may be ethanol or methanol. The salt is preferably alyotropic salt. The lyotropic salt may be selected from the groupconsisting of ammonium sulphate, sodium phosphate, and potassiumphosphate.

In one further (alternatively or additionally) preferred embodiment, themethod further comprises the step of:

-   (c) separating the particle by phase separation.

After phase separation, the produced particles can be further separatedby routine methods such as centrifugation, sedimentation, and/orfiltration.

The particle size may be optimized by adjusting one or more of thefollowing parameters: silk polypeptide and/or salt concentration,temperature during preparation, mixing intensity, and/or organic oraqueous solvents used for protein precipitation. Alternatively oradditionally, the particle size may be optimized by adjusting thegeometry of the mixing device like stirrer, static mixer or the like.

The prepared particles may subsequently be washed. The particles mayalso be stored, for example, in a dried or lyophilized form. Theprocesses may be carried out under aseptic conditions.

The particle is preferably a nanoparticle, because cellular uptake is amajor aspect of its action path and, therefore, desirable.

In one alternative embodiment, the article is a fiber. In this case, theaqueous solution may also be named spinning solution. It is preferredthat the fiber is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that the fiber issterilizable. When the article is a fiber, step (b) comprises drawing afiber from the aqueous solution/spinning solution, or extruding anddrawing a fiber from the aqueous solution/spinning solution. Thus, inone preferred embodiment, the method is for producing a fiber andcomprises the steps of:

-   (a) providing an aqueous solution comprising the polypeptide of the    first aspect, and-   (b) forming a fibre out of/from the solution provided in (a),    wherein said formation comprises drawing a fiber from the aqueous    solution/spinning solution, or extruding and drawing a fiber from    the aqueous solution/spinning solution.

Spinning methods such as wet spinning or electrospinning methods areknown to the skilled person. For example, the aqueous solution/spinningsolution is extruded through a spinneret to form a fiber. The resultingfiber can further be drawn or stretched. Whenever both crystalline andamorphous arrangements of the molecules exist in fibers, drawing orstretching will apply shear stress sufficient to orient the molecules tomake them more parallel to the walls of the fiber and increase thetensile strength and toughness of the fiber.

The fiber may be used to make a fabric, e.g. a woven or non-wovenfabric. The skilled person is aware of techniques allowing to generate afabric, e.g. weaving processes. Thus, in an alternative embodiment, thearticle may be a fabric made of fibers. The fibers may also be part of arod. Preferably, the rod comprises or consists of fibers. Bundles ofrods may also be encompassed by the present invention.

In one alternative embodiment, the article is a film. In this case, theaqueous solution may also be named casting solution. It is preferredthat the film is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that the film is sterilizable.When the article is a film, step (b) comprises casting an aqueoussolution/a casting solution comprising the polypeptide onto a substrate.Thus, in one preferred embodiment, the method is for producing a filmand comprises the steps of:

-   (a) providing an aqueous solution comprising the polypeptide of the    first aspect, and-   (b) forming a film out of/from the solution provided in (a), wherein    said formation comprises casting an aqueous solution/a casting    solution comprising the polypeptide onto a substrate.

In one further (alternatively or additionally) preferred embodiment, themethod further comprises the step of:

-   (c) separating/removing the film from the substrate.

In one alternative embodiment, the article is a capsule. It is preferredthat the capsule is not cytotoxic and not immunogenic. It is further(alternatively or additionally) preferred that the capsule issterilizable.

When the article is a capsule, step (b) comprises generating an emulsionof at least two phases, said emulsion containing the solution providedin (a) as a first phase and at least one further phase, which issubstantially immiscible with said first phase, and forming a polymernetwork of the polypeptide at the interface of the at least two phases.Thus, in one preferred embodiment, the method is for producing a capsuleand comprises the steps of:

-   (a) providing an aqueous solution comprising the polypeptide of the    first aspect, and-   (b) forming a capsule out of/from the solution provided in (a),    wherein said formation comprises generating an emulsion of at least    two phases, said emulsion containing the solution provided in (a) as    a first phase and at least one further phase, which is substantially    immiscible with said first phase, and forming a polymer network of    the polypeptide at the interface of the at least two phases.

In one further (alternatively or additionally) preferred embodiment, themethod further comprises the step of:

-   (c) separating the protein polymer network (capsule) from the    emulsion. This can be done by centrifugation, sedimentation, and/or    filtration.

A preferred method for producing capsules form the polypeptide isdescribed in WO 2007/014755 A1.

The article may also be a granule or gel. A preferred method forproducing gels from the polypeptide is described in WO 2007/014755 A1.

In a sixth aspect, the present invention relates to a pharmaceuticalcomposition comprising the article of the fourth aspect.

The article may also be named antigen carrier or antigen carriervehicle.

The article may be selected from the group consisting of a particle,capsule, fiber, and film. It is preferred that said article is notcytotoxic and not immunogenic. It is further (alternatively oradditionally) preferred that said article is sterilizable.

The inventors of the present patent application surprisingly found thatafter systemic administration of the articles, article accumulation inthe local lymph nodes and/or in antigen presenting cells, in particularin dendritic cells and/or macrophages, occurred. In addition, thearticles formed a depot under the skin. The antigen carried by thearticle induced an immune response in said cells.

Said pharmaceutical composition is useful for treating, preventing, orreducing the severity of a disease or disorder. It may be administeredlocally or systemically. It is preferred that the pharmaceuticalcomposition is formulated for local administration or systemicadministration. In particular, the local administration is by parenteraladministration, e.g. by intravenous administration, subcutaneousadministration, intradermal administration, intramuscularlyadministration, and the systemic administration is by intraarterialadministration. Preferably the composition is administeredsubcutaneously, intradermally, or intramuscularly.

It is further preferred that the composition further comprises one ormore pharmaceutically acceptable carriers, diluents, and/or excipients.An adjuvant may additionally be present.

The inventors of the present patent application surprisingly found thatthe use of adjuvants in a pharmaceutical composition comprising antigenswhich are part of articles, e.g. particles, comprising silk polypeptidesis no longer required. The effectiveness of an antigen comprised in acomposition without adjuvants is better or at least as good as the sameantigen comprised in a composition with adjuvants. The omission ofadjuvants has the positive effect that side effects and vaccineincompatibility can be reduced, in some cases even avoided. Thus, it isalternatively preferred that the composition does not further comprisean adjuvant.

In a seventh aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of the sixth aspect foruse as a pharmaceutical. The pharmaceutical may be a vaccine, e.g. forinducing an immune response or for immune therapy such as immunizationor vaccination, or an anti-cancer medicament.

In an eight aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of the sixth aspect forinducing an immune response.

It is preferred that an immune response against cancer is induced.

The cancer can be, for example, carcinoma, lymphoma, blastoma, sarcoma,or leukemia. More particularly, the cancer can be, for example, bonecancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, colon cancer, breast cancer, prostate cancer,uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, or pituitary adenoma.

It is further (alternatively or additionally) preferred that an immuneresponse against an infectious disease or autoimmune disease is induced.

An infectious disease can be, for example, a viral disease, a bacterialdisease, or a parasitic disease. More particularly, an infectiousdisease can be, for example, hepatitis, sexually transmitted diseases(e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immunedeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C,cholera, severe acute respiratory syndrome (SARS), the bird flu, orinfluenza.

An autoimmune disease can be, for example, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma, orpsoriasis.

The induction of an immune response may result in the immunization orvaccination of the treated subject/patient.

In a ninth aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of the sixth aspect foruse in a prophylactic and/or therapeutic treatment of a disease.

It is preferred that the disease is cancer.

The cancer can be, for example, carcinoma, lymphoma, blastoma, sarcoma,or leukemia. More particularly, the cancer can be, for example, bonecancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, colon cancer, breast cancer, prostate cancer,uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, or pituitary adenoma.

It is further (alternatively or additionally) preferred that the diseaseis an infectious disease or an autoimmune disease.

An infectious disease can be, for example, a viral disease, a bacterialdisease, or a parasitic disease. More particularly, an infectiousdisease can be, for example, hepatitis, sexually transmitted diseases(e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immunedeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C,cholera, severe acute respiratory syndrome (SARS), the bird flu, orinfluenza.

An autoimmune disease can be, for example, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma, orpsoriasis. It is further preferred that that the disease is arespiratory disease.

The antigen, as described herein, is preferably directly applied to thetarget area. The direct application of the antigen to the target areaallows higher local active pharmaceutical ingredients (API)concentrations while at the same time adverse side effects of a systemicapplication are minimized. The use of nebulized articles (particles,microparticles or capsules) can improve API deposition in the lowerairways. The pulmonary antigen delivery or pulmonary vaccination allowsto systemically induce immune response, because a lot of immune cellsare located in the mucosa of oral, nasal or pulmonary tissue. Theadministration of articles (particles, microparticles or capsules) tothe lung is an attractive delivery attempt for pulmonary vaccination,because the peptide is protected by the particles and will be releasedonce the particles have been taken up by antigen presenting cells. Asthere is always a risk to lose loaded antigen during the aerosolizationprocess, the articles of the fourth aspect, the pharmaceuticalcomposition of the sixth aspect, or the articles or pharmaceuticalcomposition used according to the seventh to ninth aspect are apromising alternative. Due to the integration of the activepharmaceutical part into the amino acid sequence of the antigen carriermolecule, the risk of antigen loss during nebulization is very low.

In a further aspect, the present invention relates to the article of thefourth aspect or the pharmaceutical composition of the sixth aspect forstimulating, priming, and/or expanding T cells in a subject.

In a tenth aspect, the present invention relates to a method fordelivering an antigen to a cell comprising administering to a subjectthe article of the fourth aspect or the pharmaceutical composition ofthe sixth aspect.

In a preferred embodiment, the administering comprises internalizationof the article into the cell and releasing the antigen from the article.It is preferred that the antigen is released from the article by anenzyme, which cuts the enzymatically cleavable linker (within the cell,intracellularly), thereby delivering the antigen to the cell.Preferably, the enzymatically cleavable linker is a protease cleavablelinker, more preferably a cathepsin cleavable linker, even morepreferably a cathepsin S or B cleavable linker, most preferably (i) acathepsin S cleavable linker having the sequence according to SEQ ID NO:1 or a variant thereof, or a cathepsin S cleavable linker having thesequence according to SEQ ID NO: 2 or a variant thereof, or (ii) acathepsin B cleavable linker having the sequence according to SEQ ID NO:2. The enzyme is preferably a protease, more preferably cathepsin, evenmore preferably cathepsin S or B.

It is preferred that the cell is an antigen presenting cell. It is morepreferred that the antigen presenting cell is a dendritic cell and/or amacrophage.

The subject may be healthy and the antigen is delivered for immunizationor vaccination. The subject may also be diseased (i.e. a patient), e.g.suffering from cancer, and the antigen is delivered for curing thedisease.

In an eleventh aspect, the present invention relates to a method forinducing an immune response in a subject comprising administering to asubject the article of the fourth aspect or the pharmaceuticalcomposition of the sixth aspect.

It is preferred that an immune response against cancer is induced.

The cancer can be, for example, carcinoma, lymphoma, blastoma, sarcoma,or leukemia. More particularly, the cancer can be, for example, bonecancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, colon cancer, breast cancer, prostate cancer,uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, or pituitary adenoma.

It is further (alternatively or additionally) preferred that an immuneresponse against an infectious disease or autoimmune disease is induced.

An infectious disease can be, for example, a viral disease, a bacterialdisease, or a parasitic disease. More particularly, an infectiousdisease can be, for example, hepatitis, sexually transmitted diseases(e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immunedeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C,cholera, severe acute respiratory syndrome (SARS), the bird flu, orinfluenza.

An autoimmune disease can be, for example, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma, orpsoriasis.

The induction of an immune response may result in the immunization orvaccination of the treated subject/patient.

In a twelfth aspect, the present invention relates to a method forprophylactic and/or therapeutic treatment of a disease in a subjectcomprising administering to a subject the article of the fourth aspector the pharmaceutical composition of the sixth aspect.

It is preferred that the disease is cancer.

The cancer can be, for example, carcinoma, lymphoma, blastoma, sarcoma,or leukemia. More particularly, the cancer can be, for example, bonecancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, colon cancer, breast cancer, prostate cancer,uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, or pituitary adenoma.

It is further (alternatively or additionally) preferred that the diseaseis an infectious disease or an autoimmune disease.

An infectious disease can be, for example, a viral disease, a bacterialdisease, or a parasitic disease. More particularly, an infectiousdisease can be, for example, hepatitis, sexually transmitted diseases(e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immunedeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C,cholera, severe acute respiratory syndrome (SARS), the bird flu, orinfluenza.

An autoimmune disease can be, for example, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma, orpsoriasis.

In a thirteenth aspect, the present invention relates to a method forstimulating, priming, and/or expanding T cells in a subject comprisingadministering to a subject the article of the fourth aspect or thepharmaceutical composition of the sixth aspect.

The present invention is summarized as follows:

-   1. A polypeptide comprising    -   (i) a silk polypeptide and    -   (ii) an antigen.-   2. The polypeptide of item 1, wherein the polypeptide further    comprises an enzymatically cleavable linker.-   3. The polypeptide of item 2, wherein the antigen is connected to    the silk polypeptide via the enzymatically cleavable linker.-   4. The polypeptide of items 2 or 3, wherein the enzymatically    cleavable linker is a protease cleavable linker.-   5. The polypeptide of item 4, wherein the protease cleavable linker    is a cathepsin cleavable linker.-   6. The polypeptide of item 5, wherein the cathepsin cleavable linker    is a cathepsin S cleavable linker or a cathepsin B cleavable linker.-   7. The polypeptide of item 6, wherein    -   (i) the cathepsin S cleavable linker has the sequence according        to SEQ ID NO: 1 or is a variant thereof, or SEQ ID NO: 2 or is a        variant thereof, or    -   (ii) the cathepsin B cleavable linker has the sequence according        to SEQ ID NO: 2 or is a variant thereof.-   8. The polypeptide of any one of items 1 to 7, wherein the silk    polypeptide is a recombinant silk polypeptide.-   9. The polypeptide of any one of items 1 to 8, wherein the silk    polypeptide comprises at least two identical repetitive units.-   10. The polypeptide of item 9, wherein the repetitive units are    independently selected from the group consisting of module C having    the sequence according to SEQ ID NO: 3 or a variant thereof, module    C^(Cys) having the sequence according to SEQ ID NO: 4, and module    C^(kappa) having the sequence according to SEQ ID NO: 18.-   11. The polypeptide of any one of items 1 to 10, wherein the silk    polypeptide comprises at least one non-repetitive (NR) unit and/or    Tag.-   12. The polypeptide of any one of items 1 to 11, wherein the antigen    is selected from the group consisting of a viral antigen, a    microbial antigen, preferably a bacterial antigen or a fungal    antigen, a zooparasitic antigen, and a tumor antigen.-   13. A nucleic acid molecule encoding the polypeptide of any one of    items 1 to 12.-   14. A method for producing a polypeptide comprising the step of:    -   (a) expressing the nucleic acid molecule of item 13 in a cell,        thereby producing the polypeptide in the cell.-   15. The method of item 14, wherein the method further comprises the    step of:    -   (b) isolating the polypeptide from the cell.-   16. An article comprising the polypeptide of any one of items 1 to    12.-   17. The article of item 16, wherein the article is selected from the    group consisting of a particle, capsule, fiber, film, granule, gel,    fabric made of fibers, rod or bundles thereof-   18. The article of item 17, wherein the particle has an average    diameter in the range of from 50 nm to 1000 nm.-   19. The article of any one of items 17 or 18, wherein the particle    has a net negative surface charge.-   20. The article of any one of items 17 to 19, wherein the particle    is not cytotoxic and not immunogenic.-   21. The article of any one of items 17 to 20, wherein the particle    is sterilisable.-   22. A method for producing an article comprising the steps of:    -   (a) providing an aqueous solution comprising the polypeptide of        any one of items 1 to 12, and    -   (b) forming an article out of/from the solution provided in (a).-   23. The method of item 22, wherein the concentration of the    polypeptide in the aqueous solution is of between 0.1 wt %/vol and    30 wt %/vol, preferably between 1 wt %/vol and 20 wt %/vol.-   24. The method of items 22 or 23, wherein the article is a particle    and wherein step (b) comprises triggering aggregation of the    polypeptide.-   25. The method of item 24, wherein aggregation is triggered by pH    shift, ion exchange, shear forces, the addition of alcohol, the    addition of a salt, preferably a lyotropic salt, the removal of    water, temperature change, and by combinations thereof-   26. The method of item 25, wherein    -   (i) the alcohol is ethanol or methanol, or    -   (ii) the lyotropic salt is selected from the group consisting of        ammonium sulphate, sodium phosphate, and potassium phosphate.-   27. The method of any one of items 24 to 26, wherein the method    further comprises the step of:    -   (c) separating the particle by phase separation.-   28. A pharmaceutical composition comprising the article of any one    of items 16 to 21.-   29. The pharmaceutical composition of item 28, wherein the    composition further comprises one or more pharmaceutically    acceptable carriers, diluents and/or excipients.-   30. The pharmaceutical composition of items 28 or 29, wherein the    composition further comprises an adjuvant.-   31. The pharmaceutical composition of items 28 or 29, wherein the    composition does not further comprise an adjuvant.-   32. The article of any one of items 16 to 21 or the pharmaceutical    composition of any one of items 28 to 31 for use as a    pharmaceutical.-   33. The article of any one of items 16 to 21 or the pharmaceutical    composition of any one of items 28 to 31 for inducing an immune    response.-   34. The article of any one of items 16 to 21 or the pharmaceutical    composition of any one of items 28 to 31 for use in a prophylactic    and/or therapeutic treatment of a disease.-   35. A method for delivering an antigen to a cell comprising    administering to a subject the article of any one of items 16 to 21    or the pharmaceutical composition of any one of items 28 to 31.-   36. The method of item 35, wherein the cell is an antigen presenting    cell.-   37. The method of item 36, wherein the antigen presenting cell is a    dendritic cell and/or a macrophage.-   38. A method for inducing an immune response in a subject comprising    administering to a subject the article of any one of items 16 to 21    or the pharmaceutical composition of any one of items 28 to 31.-   39. A method for prophylactic and/or therapeutic treatment of a    disease in a subject comprising administering to a subject the    article of any one of items 16 to 21 or the pharmaceutical    composition of any one of items 28 to 31.-   40. A method for stimulating, priming, and/or expanding T cells in a    subject comprising administering to a subject the article of any one    of items 16 to 21 or the pharmaceutical composition of any one of    items 28 to 31.

The sequence listing comprises the following sequences:

-   SEQ ID NO: 1 cathepsin S cleavable linker-   SEQ ID NO: 2 cathepsin S and B cleavable linker-   SEQ ID NO: 3 module C-   SEQ ID NO: 4 module C^(Cys)-   SEQ ID NO: 5 T7-Tag-   SEQ ID NO: 6 T7-Tag-   SEQ ID NO: 7 NR3 unit-   SEQ ID NO: 8 NR4 unit-   SEQ ID NO: 9 NR5 unit-   SEQ ID NO: 10 NR6 unit-   SEQ ID NO: 11 epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄), SIINFEKL-   SEQ ID NO: 12 C16-   SEQ ID NO: 13 C16-SIINFEKL-   SEQ ID NO: 14 C16-CathS-SIINFEKL-   SEQ ID NO: 15 C16-CathB-SIINFEKL-   SEQ ID NO: 16 IGSIINFEKLG sequence cleaved from the hybrid    polypeptide comprising the cathepsin B cleavable linker and the    epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄)-   SEQ ID NO: 17 LPGSIINFEKLG sequence cleaved from the hybrid    polypeptide comprising the cathepsin S cleavable linker and the    epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄)-   SEQ ID NO: 18: module C^(kappa)

Regarding SEQ ID NO: 12 to SEQ ID NO: 15 it should be noted that theC-terminal G (Gly) may be present or not.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope ofinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artin the relevant fields are intended to be covered by the presentinvention.

BRIEF DESCRIPTION OF THE FIGURES

The following figures and examples are merely illustrative of thepresent invention and should not be construed to limit the scope of theinvention as indicated by the appended claims in any way.

FIGS. 1A-1D: None of the spider silk particles induce BMDC cytotoxicityin vitro

FIG. 1A shows a dot plot from flow cytometry with propidium iodide (PI)and annexin V. BMDC (5×10⁴ cells/well) were cultured with spider silkparticles at 505 ug particle/mL (=10 ug SIINFEKL (SEQ ID NO: 11)/mL).After 24 hours of incubation, BMDC viability was assessed by flowcytometry and MTT assay.

(FIG. 1A) Representative dot plot from flow cytometry with propidiumiodide (PI) and annexin V.

(FIG. 1B) Scheme of the gating strategy to quantify live healthy cells(annexin V−/PI−).

(FIG. 1C) Percentage of live healthy cells (annexin V−/PI−).

(FIG. 1D) Optical density (OD) at 570 nm correlating with formazanproduction from the MTT assay.

Condition without cells (medium only) was used as control. n.d.: notdone. Asterisks (****, P<0.0001) indicate significant differences withuntreated control group using one-way ANOVA followed by Dunnett'smultiple comparison test. (FIG. 1C and FIG. 1D) Each bar representsmean±SEM of 3 independent experiments performed in duplicate. (Exceptfor Panel C, C16-SIIN: tested once in duplicate). Medium only was usedas control, untreated: untreated control group; SIIN: SIINFEKL (SEQ IDNO: 11) peptide alone; C16: native C16 particles; C16-SIIN;C16-CathBseq-SIIN, C16-CathSseq-SIIN hybrid protein particles.

FIGS. 2A-2B: The spider silk particles do not induce BMDC immunologicalactivation in vitro

FIG. 2A shows the median fluorescent intensity (MFI) of BMDC surfaceactivation markers MHC I and MHC II of BMDC cell cultured with SIIN,CathBseq-SIIN and C16-CathSseq-SIIN compared to untreated sample. FIG.2B shows the Cytokine quantification with ELISA. R848 (R8), a TLR7agonist, was used as positive control. BMDC (5×10⁴ cells/well) werecultured with spider silk particles at 50 ug particle/mL. After 24 hoursof incubation, BMDC were analysed by flow cytometry, whereas supernatantwas collected for cytokine quantification.

(FIG. 2A) Median fluorescent intensity (MFI) of BMDC surface activationmarkers: fold change compared to untreated sample.

(FIG. 2B) Cytokine quantification with ELISA. R848 (R8), a TLR7 agonist,was used as positive control (0.25 ug/mL). Asterisks (***, P<0.001)indicate significant differences with untreated control group usingone-way ANOVA followed by Dunnett's multiple comparison test. Each barrepresents mean±SEM of 4 independent experiments performed in duplicate.

FIGS. 3A-3B: Spider silk particles are efficiently taken up byantigen-presenting cells FIG. 3A shows the uptake of C16 particleswithout SIIN (C16), C16 hybrid particles C16-SIIN, C16 hybrid particleswith Cathepsin B cleavage site (C16-CathBseq-SIIN) and C16 hybridparticles with Cathepsin S cleavage site (C16-CathSseq-SIIN). Untreatedcells (untreated) and SIIN polypeptide (SIIN) serve as controls. FIG. 3Bshows the percentage of FITC-positive cells determined in defined immunecell populations: T cells (CD3+), dendritic cells (CD11c+CD11b+) andmonocytes/macrophages (CD11c-CD11b+) compared to untreated cells.

(FIG. 3A) BMDC (5×10⁴ cells/well) were cultured with FITC-labelledspider silk particles at 50 ug particles/mL. After 24 hours ofincubation, BMDC were isolated for flow cytometry analysis. Percentageof FITC-positive cells within BMDC (CD11c+) population was determined.Each bar represents mean±SEM of 2 independent experiments performed induplicate. (Except for C16-SIIN: tested once in duplicate).

(FIG. 3B) Freshly isolated splenocytes (5×10⁴ cells/well) were cultured6 hours with FITC-labelled spider silk particles. After 6 hours ofincubation, cells were analysed by flow cytometry. Percentage ofFITC-positive cells was determined in defined immune cell populations: Tcells (CD3+), dendritic cells (CD11c+CD11b+) and monocytes/macrophages(CD11c-CD11b+). Graph depicts one representative experiment of 3. Eachexperiment was performed in duplicate.

FIG. 4 : The Cathepsin S sequence is the most effective to induceSIINFEKL (SEQ ID NO: 11) dependent in vitro T-cell proliferation

FIG. 4 shows the percentage of proliferating CD8 T-cells within the Tcell population (CD3+CD8+). The percentage of proliferating of CD8T-cells exposed to BMDC cells with C16 CathSseq-SIINFEKL particles andC16 CathBseq-SIINFEKL particles was significantly higher compared to theuntreated control. The percentage of proliferating of CD8 T-cellsexposed to BMDC cells with C16 CathSseq-SIINFEKL particles was furthersignificantly higher than the percentage of exposed to BMDC cells withC16 CathBseq-SIINFEKL particles. BMDC (5×104 cells/well) were culturedwith spider silk particles at 50 ug particles/mL. R848 (0.25 ug/mL) wasused as adjuvant for BMDC activation. After 24 hours of incubation, CFSElabelled CD3+CD8+ OT-I cells (10⁵ cells/well) were added. After 3 daysof co-culture, the cells were analyzed by flow cytometry.

Percentage of proliferating cells within the T cell population(CD3+CD8+). Each bar represents mean±SEM of 2 independent experimentsperformed in quadruplicate. Asterisks (**, P<0.01) indicate significantdifferences between R848-treated groups using two-way ANOVA followed byTukey's multiple comparison test.

FIGS. 5A-5B: Hybrid spider silk particles accumulate in the draininglymph node in vivo.

FIG. 5A shows the number of FITC positive cells (comprisingFITC-labelled C16-CathSseq-SIINFEKL particles) in draining lymph nodes(DLN) of 3 FITC-particle treated mice compared to a control group of 3PBS treated mice in several tissues (DLN, non-DLN and spleen).FITC-labelled C16-CathSseq-SIINFEKL particles were injectedsubcutaneously into the right flank of 3 mice (505 ug particles in 1004,PBS per mouse). PBS was used as negative control. After 24 hours, theipsilateral draining lymph nodes (DLN), the contralateral lymph nodes(non DLN) and the spleen were isolated for flow cytometry analysis.

(FIG. 5A) Number of FITC-positive cells in the different lymphaticorgans

(FIG. 5B) Percentage of FITC-positive cells within defined immune cellpopulations. Each dot represents one mouse. Bars represent mean±SEM.Asterisks (***, P<0.001) indicate significant differences when comparingFITC-particle treated mice with PBS-treated mice using two-way ANOVAfollowed by Bonferroni's multiple comparison test.

FIG. 5A shows that C16-CathSseq-SIINFEKL particles accumulated in thedraining lymph node (DLN) after administration. The results illustratedin FIG. 5B point towards an uptake by dendritic cells (CD11c+CD11b+)rather than by macrophages (CD11c−CD11b+) or leukocytes (CD11b−).

FIG. 6 : SIINFEKL (SEQ ID NO: 11)-containing spider silk particlesinduce antigen-dependent T-cell proliferation in vivo.

FIG. 6 shows the proliferation of CD8 T cells of mice immunized withC16-CathSseq-SIINFEKL particles. The proliferation of CD8 T cells wassignificantly higher without R848 adjuvant.

10⁶ CFSE-labelled CD3+CD8+ OT-I cells in 100 μL of PBS were injectedintravenously into mice (each dot represents one mouse). 18 hours later,mice were vaccinated with spider silk particles (505 ug particles in 100μL PBS per mouse). R848 (25 ug) was used as adjuvant. 3 days aftervaccination, the DLN were isolated for flow cytometry analysis todetermine the proliferation of CD3+CD8+ CFSE-labelled OT-I cells. Eachdot represents one mouse. Bars represent mean±SEM. Asterisks (****,P<0.0001) indicate significant differences with R848-treated controlusing one-way ANOVA followed by Dunnett's multiple comparison test. Theproliferation of CD8 T cells was higher without R848 adjuvant. It couldbe shown that the C16-CathSseq-SIINFEKL protein particles were able toinduce similar to respectively higher level of proliferating CD8+ Tcells either with, but also without the use of the immunostimulatoryadjuvant R848.

FIGS. 7A-7B: Cathepsin S enzyme Incubation of C16 particlesC16-CathB-SIINFEKL and C16-CathS-SIINFEKL with cathepsin S and cathepsinB enzymes in vitro Differential release of the antigen with differentcathepsin enzymes. The sequence LPGSIINFEKLG (SEQ ID NO: 17) wasreleased from C16-CathS-SIINFEKL hybrid protein particles, while thesequence IGSIINFEKLG (SEQ ID NO: 16) was released fromC16-CathB-SIINFEKL hybrid protein particles. Said sequences comprise thesequence SIINFEKL (SEQ ID NO: 11). Data are the mean and SD (standarddeviation) of 3 independent replicates. Cathepsin S enzyme shows thebetter cleavage of SIINFEKL (SEQ ID NO: 11) peptides from both C16hybrid protein particles than cathepsin B. Cathepsin S also cleavesSIINFEKL (SEQ ID NO: 11) peptides from particles designed for cathepsinB release.

(FIG. 7A) eADF4(C16) hybrid protein particles incubated with cathepsin Senzyme for 96 hours.

(FIG. 7B) eADF4(C16) hybrid protein particles incubated with cathepsin Benzyme for 96 hours.

EXAMPLES

The examples given below are for illustrative purposes only and do notlimit the invention described above in any way.

The terms “NP” and “spider silk particle”, the terms “SIIN” and“SIINFEKL”, the terms “C16-CathB-SIINFEKL”, “C16-CathB-SIIN”,“eADF4(C16) CathB” and “SSP25-eADF4(C16-CathB-CD8)”, the terms“C16-CathS-SIINFEKL”, “C16-CathS-SIIN”, “eADF4(C16) CathS” andSSP26-eADF4(C16-CathS-CD8)” as well as the terms “SIIN” and “SIINFEKL”are used interchangeably herein. SIINFEKL (SEQ ID NO: 11) represents theamino acid sequence of an epitope of chicken-Ovalbumin (OVA₂₅₇₋₂₆₄).This antigen stimulates an immune response via interaction with MHC Iand CD8 T-cell receptor.

Example 1: Production of Silk Polypeptide Antigen Particles

The polypeptides SSP25-eADF4(C16-CathS-CD8) (1),SSP26-eADF4(C16-CathB-CD8) (2), C16-SIIN (3) and C16 (4) weresynthesized via gene syntheses at Geneart (Regensburg).

-   (1) C16-CathS-SIINFEKL respectively SSP26-eADF4(C16-CathS-CD8) (SEQ    ID NO: 14)    -   (C16) GPMGLPG SIINFEKL    -   Hybrid polypeptide comprising C16, a Cathepsin S protease        cleavage site, and SIINFEKL (SEQ ID NO: 11).-   (2) C16-CathB-SIINFEKL respectively SSP25-eADF4(C16-CathB-CD8) (SEQ    ID NO: 15)    -   (C16) GAVGFLGIG SIINFEKL    -   Hybrid polypeptide comprising C16, a Cathepsin B protease        cleavage site, and SIINFEKL (SEQ ID NO: 11).-   (3) C16-SIINFEKL (SEQ ID NO: 13)    -   (C16) GGSG SIINFEKL    -   Hybrid polypeptide comprising C16 and SIINFEKL (SEQ ID NO: 11).-   (4) C16 (SEQ ID NO: 12)    -   C16    -   Hybrid polypeptide without antigen (SIINFEKL (SEQ ID NO: 11)).

The polypeptides encoding SSP25-eADF4(C16-CathB-CD8),SSP26-eADF4(C16-CathS-CD8), C16-SIINFEKL and C16 were produced andpurified as described in WO 2006/008163 A2.

Example 2: Sterilization by Autoclave Treatment

In a first step, about 150 mg of the protein (C16-CathB-SIINFEKL,C16-CathS-SIINFEKL, C16-SIINFEKL and C16) were weighed into glass vials(DIN 10R). The C16 protein was subsequently suspended with 7.5 ml HPW(highly purified water). The vials were closed with rubber stoppers andcrimped with aluminum caps. Steam sterilization was performed for 15minutes at 121° C. in a GTA 50 autoclave (Fritz Gössner, Hamburg,Germany). After cooling down, the C16 protein suspension was centrifugedat 10,000 rpm (SIGMA 4K15, Sigma Laborzentrifugen, Osterode am Harz,Germany) for 30 minutes and the supernatant was discarded. Thecentrifuged C16 protein was dissolved in a 6 M guanidine thiocyanatesolution and dialyzed against an endotoxin free 10 mM TRIS/HCl solutionpH 8.0 for 24 h.

The protein concentration of C16-CathB-SIINFEKL after dialysis was 3.91mg/ml, the protein concentration of C16-CathS-SIINFEKL was 3.56 mg/ml,the protein concentration of C16-SIINFEKL was 3.81 mg/ml and the proteinconcentration of C16 was 3.88 mg/ml. The protein concentration of allsolutions was adjusted to 1 mg/ml. The endotoxin values of the solutionswere <0.200 EU/mg. Sterilization of spider silk particles had nodetrimental effect on particle size, secondary structure and thermalstability. After sterilization by autoclave treatment, no changes insize or secondary structure of the particles as well as no functionalchanges were observed. Remaining the function after sterilization isadvantageous in view of the systems of the prior art.

Example 3: Particle Preparation

After endotoxin removal, the protein solutions (C16-CathB-SIINFEKL,C16-CathS-SIINFEKL, C16-SIINFEKL and C16) were adjusted to 1 mg/ml withendotoxin free 10 mM TRIS/HCl buffer pH 8.0 for particle preparation.The particle preparation was carried out by micromixing using a highpressure syringe pump system. The syringe pump cylinders weredepyrogenized by 70% (v/v) ethanol over 48 h. Subsequently, thecylinders were washed three times with HPW to remove any organicsolvent. After depyrogenation, both cylinders of the syringe pump system(Model 100 DX and Series D pump controller, Teledyne Isco, Lincoln, USA)were filled with pre-tempered C16 solution and pre-tempered endotoxinfree 2 M potassium phosphate buffer pH 8.0 or 4 M ammonium sulfatesolution of 80°. The solutions were pumped at a high flow rate of 50ml/min to a T-shape mixing element (inner diameter 0.5 mm, P-727 PEEKtee, Upchurch Scientific, Oak Harbor, USA) leading to an outlet tubing(inner diameter 0.5 mm, 1532 PEEK Tubing, Upchurch Scientific, OakHarbor, USA) for suspension collection. The C16 particle suspensionswere subsequently centrifuged at 14,000 rpm (SIGMA 4K15, SigmaLaborzentrifugen, Osterode am Harz, Germany) and washed with HPW threetimes. A two minute ultrasonication (Sonopuls HD 3200, Bandelinelectronic, Berlin, Germany) step completed the particle preparationprocedure. The particle concentrations in mg/ml were determinedgravimetrically after drying the particles under vacuum (13 mbar)overnight.

The particle concentration of C16-CathB-SIINFEKL was 32.75 mg/ml, theprotein concentration of C16-CathS-SIINFEKL was 34.72 mg/ml, the proteinconcentration of C16-SIINFEKL was 30.06 mg/ml and the proteinconcentration of C16 was 32.52 mg/ml.

Example 4: Optimizing C16 Particle Size

The micromixing particle preparation process was analyzed for furtherreduction of the final particle size. Some parameters were selected tobe changed compared to the preparation process above. The concentrationof the C16 solution used for particle preparation was adjusted to0.5-1.0 mg/ml. The 2 M potassium phosphate solution used for particleprecipitation so far was complemented by a 2 M, a 3 M and a 4 M ammoniumsulfate solution. The flow rate of the salt solution was kept at 50ml/min, whereas the flow rate of the protein solution was set to 25-50ml/min. For this studies, native C16 protein was used. All otherparameters were kept as described above and final particle size wasanalyzed after particle preparation with the modified syringe pumpsettings described here. The resulting particles have an averagediameter in the range of from 250 nm to 520 nm

Example 5: In Vitro Release of SIINFEKL (SEQ ID NO: 11) from Hybrid C16Particles (In Vitro)

The release of the antigen sequence SIINFEKL (SEQ ID NO: 11) from thehybrid polypeptides SSP25-eADF4(C16-CathB-CD8) andSSP26-eADF4(C16-CathS-CD8) was tested by the addition of cathepsinenzymes. As the hybrid polypeptides contain cleavable linker sequencesfor the cathepsin S and cathepsin B enzymes, these two cathepsins werealso used for the in vitro release studies.

The C16 hybrid polypeptide particles were suspended to a finalconcentration of 2 mg/ml with a 50 mM sodium acetate buffer, pH 5.5,containing 1 mM EDTA and 2 mM DTT for incubation with the cathepsin Senzyme. Cathepsin S was diluted in the same buffer to a finalconcentration of 0.9 mU/ml. Slight pH modification of the buffer wasrealized for the cathepsin B enzyme.

The C16 hybrid polypeptide particles were suspended to a finalconcentration of 2 mg/ml with a 50 mM sodium acetate buffer, pH 5.0,containing 1 mM EDTA and 2 mM DTT for incubation with the cathepsin Benzyme. Cathepsin B was diluted in the same buffer to a finalconcentration of 0.1 U/ml. The incubation of the particles with theenzymes was carried out at 37° C. on a waving platform shaker (HeidolphPolymax 1040, Heidolph Instruments GmbH, Schwabach, Germany) at 10 rpm.Samples (supernatant) were drawn after 1, 6, 24, 48, 72 and 96 h andused for analysis by RP-HPLC.

The cleaved SIINFEKL (SEQ ID NO: 11) peptide fragments were analyzed byRP-HPLC. The supernatant of each sample was removed from the particlesby centrifugation (two times at 12,000 rpm for 30 minutes). The pelletswere discarded and 180 μl of the supernatant was filled into HPLC glassinserts and analyzed by RP-HPLC (detection by UV-Vis at 220 nm). Volumesof 50 μl of the corresponding supernatants were separated at 30° C. by areversed phase YMC-Triart C18 column (YMC Europe GmbH, Dinslaken,Germany) using a Waters 2695 separations module (Waters Corporation,Milford, Mass., USA). A gradient with two mobile phases was applied,using water+0.1% [m/m] TFA (mobile phase A) and 100% acetonitrile+0.1%[m/m] TFA (mobile phase B). Each run started with two minutes of 95%mobile phase A and was followed by a linear increase of mobile phase Bfrom 5% to 100% over 28 minutes. A five minute washing step with 100%mobile phase B was used to wash residual peptide/protein from thecolumn. The separation run stopped with a five minute equilibration ofthe column at 95% mobile phase A. The detection was carried out on aWaters UV-Vis detector 2487 (Waters Corporation, Milford, Mass., USA) ata wavelength of 220 nm to detect the SIINFEKL (SEQ ID NO: 11) peptides.The amount of the released SIINFEKL (SEQ ID NO: 11) peptides wasanalyzed using a standard curve. In particular, cleaving the SIINFEKL(SEQ ID NO: 11) peptide of the hybrid polypeptide with the cathepsin Scleavable linker resulted in a peptide with the sequence IGSIINFEKLG(SEQ ID NO: 16). In addition, cleaving the SIINFEKL (SEQ ID NO: 11)peptide of the hybrid polypeptide with the cathepsin S cleavable linkerresulted in a peptide with the sequence LPGSIINFEKLG (SEQ ID NO: 17).SIINFEKL (SEQ ID NO: 11) is comprised in IGSIINFEKLG (SEQ ID NO: 16) aswell as in LPGSIINFEKLG (SEQ ID NO: 17). These two peptides were usedfor the standard curve at concentrations of 10, 20, 30, 50 and 100 μg/mldissolved in 50% DMSO/50% water. The area of each of the peptides in thechromatogram was integrated and used for calculation of calibrationcurves after injection and analysis. Data analysis was performed withChromeleon® 6.80 software (Dionex GmbH, Germering, Germany).

FIG. 7A shows the total release in percent of the cleaved SIINFEKL (SEQID NO: 11) peptides for the hybrid polypeptidesSSP25-eADF4(C16-CathB-CD8) and SSP26-eADF4(C16-CathS-CD8) as a functionof time (1, 6, 24, 48, 72 and 96 h).

Cathepsin S enzyme shows the better cleavage of SIINFEKL (SEQ ID NO: 11)peptides from both hybrid polypeptide particles. Cathepsin S alsocleaves SIINFEKL (SEQ ID NO: 11) peptides from particles designed forcathepsin B release, but slower and in a lesser extent. This could notbe expected, because the linker which was assigned for the cleavage bythe cathepsin S enzyme (PMGLP, SEQ ID NO: 20) and not for cleavage ofthe cathepsin B linker sequence (GFLG, SEQ ID NO: 21).

In contrast to Cathepsin B, Cathepsin S is only expressed in certaintissue. Cathepsin S plays a key role in the degradation of antigenicproteins and the further processing via the Major HistocompatibilityComplex Class II pathway. Cathepsin S linker particles were chosen forin-vivo mice studies.

FIG. 7 A shows the incubation of the hybrid protein particles withcathepsin S enzyme, while FIG. 7 B shows the incubation of the hybridprotein particles with cathepsin B enzyme. Enzymatic cleavage of theSIINFEKL (SEQ ID NO: 11) peptide from the SSP hybrid protein particlesis successful in vitro, which is the basis for the further in vitro andin vivo studies.

Example 6: Spider Silk Hybrid Particles do not Induce BMDC Cytotoxicity

Preparation of Bone Marrow-Derived Dendritic Cells (BMDC)

BMDC were generated from primary bone marrow cells obtained by flushingtibia and femurs of C57BL/6Rj mice with cold PBS. Red blood cells lysiswas performed with BD Pharm Lyse (BD Biosciences, USA) for 1 min. Cellswere then resuspended in complete BMDC medium consisting of RPMI 1640(Biowest, France) supplemented with 10% FCS (Biological Industries,Israel), 1% L-glutamine, 50 U/mL Penicillin, 50 U/mL Streptomycin, 50 μM2-mercaptoethanol and 0.5 mM of sodium pyruvate (all from PAALaboratories, Austria) supplemented with 40 ng/ml GM-CSF (PeproTech,USA). Loosly adherent cells were harvested after 6 days differentiation.The percentage of CD11c+CD11b+ cells was routinely over 70%.

Exposure to Spider Silk Particles

BMDC (5×10⁴ cells per well) were seeded in flat-bottom 96-well plates(Corning, N.Y., USA) in presence of C16 particles (untreated), C16hybrid particles (with SIIN polypeptide (SIIN), SEQ ID NO: 13), C16hybrid particles with Cathepsin B cleavage site (C16-CathBseq-SIIN, SEQID NO: 15) and C16 hybrid particles with Cathepsin S cleavage site(C16-CathSseq-SIIN, SEQ ID NO: 14) at 505 ug particle/mL (=10 ugSIINFEKL (SEQ ID NO: 11)/mL) (This is 10 times more than theconcentration used in other in vitro experiments) diluted in 100 μL ofcomplete medium per well. The TLR7 agonist R848 (Invitrogen, USA), wasused as immunostimulant. After 24 hours of incubation, cells wereharvested for flow cytometry analysis, whereas the supernatant wasstored for cytokine quantification. BMDC viability was assessed by flowcytometry and MTT assay.

Fluorescent Labelling

The labelling of C16-, C16-SIIN, C16-CathBseq-SIIN- andC16-CathSseq-SIIN-polypeptides with fluorescein isothiocyanate (FITC)was performed based on the published method by Spieß et al. (Spieß etal. 2010) using the terminal amine group of C16. FITC-labeled The SIINpolypeptides were obtained from GenScript Inc., Piscataway Township,N.J., USA. For the preparation of particles used for in vivo studies,the C16 protein powder dry protein suspended in HPW was autoclaved asdescribed before. After steam sterilization, the autoclaved C16 powderwas dissolved in a 6 M guanidine thiocyanate solution but this timedialyzed against an endotoxin free 20 mM HEPES solution pH 8.0 at 2-8°C. for 24 h. After dialysis, centrifugation and filtration, the solutionwas adjusted to a concentration of 2.0 mg/ml with an endotoxin free 20mM HEPES solution pH 8.0 for coupling in solution. A 20-fold molarexcess of FITC (dissolved in DMSO) was added slowly to the C16 solution.After addition of the whole amount of dissolved FITC, the solution wasincubated in the dark for three hours at room temperature. Afterincubation, the FITC coupled C16 protein solution was filtered firstwith a 0.2 μm PES filter (VWR International, Radnor, USA) andsubsequently filtered with a pre-flushed Mustang® E filter. The filteredFITC coupled C16 protein solution was adjusted to a proteinconcentration of 1 mg/ml for particle preparation by the syringe pumpsystem at 80° C. All other parameters were identical with the previouslydescribed particle preparation process. The fluorescent labelling ofparticles used only for in vitro studies was carried out at the finalparticles. The C16 particles were suspended at a concentration of 2.5mg/ml in an endotoxin free 20 mM HEPES buffer pH 8.0. A 20-fold molarexcess of FITC (dissolved in DMSO) was added dropwise to the particlesuspension. After incubation for 72 h in the dark, the particles werecentrifuged and washed with HPW for three times. Additionalultrasonication for 2 minutes finished the FITC labelling process offinal C16 polypeptide particles.

Dynamic Light Scattering (DLS)

Particle size and size distribution of submicroparticles were measuredin triplicate by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern Instruments, Worcestershire, UK). Particle size is shown asthe Z-average value, and the particle size distribution is displayed bythe polydispersity index (PDI). Directly before each measurement,samples were diluted to a final concentration of 0.01 mg/ml with HPW.All measurements were conducted at 25° C.

Cytotoxicity

Cytotoxicity was assed by flow cytometry using propidium iodide (PI) andannexin V staining and by assay. For flow cytometry analysis, BMDC wereincubated with APC-annexin V diluted 1:100 in annexin V buffer (bothfrom Biolegend, USA) for 30 min at room temperature. 0.24 PI(Sigma-Aldrich, USA) diluted 1:2 in PBS was automatically added by theMACS quant analyzer (Miltenyi Biotec, Germany) just before analysis. Allflow cytometry data were analyzed using FlowJo version 10.0.8r1.Staurosporine (1 nM) (Sigma-Aldrich, USA) was used as positive control(not shown). For the MTT assay, Vybrant MTT cell proliferation Assay Kit(Molecular Probes, USA) was used according to manufacturer protocol. Forthis assay, complete BMDC medium without phenol red (Biowest, France)was used.

BMDC Cell Phenotyping

After washing with FACS buffer consisting of PBS (Eurobio, France)supplemented with 2 mM EDTA (Calbiochem, Germany) and 0.5% BSA (PAAlaboratories, Austria), the BMDC cells were incubated with anti-mouseCD16/32 to block Fc receptors (Biolegend, USA). After 10 min incubationat 4° C., antibodies for activation markers or their respective isotypecontrols were added: PB-CD80, PE-CD86, APC-MI-ICI and FITC-CD11b (allfrom Biolegend, USA). Dead cells were excluded using zombie violet dye(Biolegend, USA). After 30 minute incubation at 4° C., the cells werewashed and resuspended in FACS buffer before acquisition.

FIG. 1A shows a dot plot from flow cytometry with propidium iodide (PI)and Annexin V. No difference in cell viability could be detected betweencells without C16 particles (untreated), SIIN polypeptide (SIIN), C16particles (C16), C16 hybrid particles with Cathepsin B cleavage site(C16-CathBseq-SIIN) and C16 hybrid particles with Cathepsin S cleavagesite (C16-CathSseq-SIIN).

FIG. 1B shows the schematic diagram of live, healthy cells, dead cellsand apoptotic cells.

FIG. 1C shows the percentage of live cells with SIIN, C16-CathBseq-SIINand C16-CathSseq-SIIN compared to untreated cells and cells with C16particles. No difference in cell viability could be detected betweencells without C16 particles (untreated), SIIN polypeptide (SIIN), C16particles (C16), C16 hybrid particles with Cathepsin B cleavage site(C16-CathBseq-SIIN) and C16 hybrid particles with Cathepsin S cleavagesite (C16-CathSseq-SIIN).

FIG. 1D shows the optical density (OD) at 570 nm correlating withformazan production from the MTT assay. There is no difference inoptical density between spider silk particles as well as the spider silkhybrid particles compared to the control (untreated). This shows thatthe spider silk particles as well as the spider silk hybrid particlesdid not induce BDMC cytotoxicity.

Example 7: Spider Silk Particles do not Induce BMDC ImmunologicalActivation In Vitro

Preparation of Bone Marrow-Derived Dendritic Cells (BMDC)

BMDC were generated from primary bone marrow cells obtained by flushingtibia and femurs of C57BL/6Rj mice with cold PBS. Red blood cells lysiswas performed with BD Pharm Lyse (BD Biosciences, USA) for 1 min. Cellswere then resuspended in complete BMDC medium consisting of RPMI 1640(Biowest, France) supplemented with 10% FCS (Biological Industries,Israel), 1% L-glutamine, 50 U/mL Penicillin, 50 U/mL Streptomycin, 50 μM2-mercaptoethanol and 0.5 mM of sodium pyruvate (all from PAALaboratories, Austria) supplemented with 40 ng/ml GM-CSF (PeproTech,USA). Loosly adherent cells were harvested after 6 days differentiation.The percentage of CD11c+CD11b+ cells was routinely over 70%.

The BMDC (5×10⁴ cells/well) were cultured with spider silk particles at50 ug particle (spider silk particle)/mL.

BMDC Phenotyping

After washing with FACS buffer consisting of PBS (Eurobio, France)supplemented with 2 mM EDTA (Calbiochem, Germany) and 0.5% BSA (PAAlaboratories, Austria), the cells were incubated with anti-mouse CD16/32to block Fc receptors (Biolegend, USA). After 10 min incubation at 4°C., antibodies for immune activation markers or their respective isotypecontrols were added: PB-CD80, PE-CD86, APC-MI-ICI and FITC-CD11b (allfrom Biolegend, USA). Dead cells were excluded using zombie violet dye(Biolegend, USA). After 30 minute incubation at 4° C., the cells werewashed and resuspended in FACS buffer before acquisition.

After 24 hours of incubation, BMDC were analysed by flow cytometry,whereas supernatant was collected for cytokine quantification.

Analysis of Cytokine Production by ELISA

ELISA Max deluxe sets for mouse IL-6 (Biolegend, USA) was used accordingto the manufacturer's protocol. For protein concentration, absorbance at570 nm was measured and subtracted from the absorbance at 450 nm byInfinite 200 PRO plate-reader (TECAN, Switzerland). Concentrations werecalculated according to the standard curve performed in duplicate.

FIG. 2A shows the median fluorescent intensity (MFI) of BMDC surfaceactivation markers MHC I and MHC II of BMDC cells cultured with SIIN,C16-SIIN, CathBseq-SIIN and C16-CathSseq-SIIN compared to untreatedsample. The adjuvant R848 (R8), a TLR7 agonist, was used as positivecontrol. No significant increase of the immune activation markers MHC Iand MHC II could be detected. This shows that the spider silk particlesas well as the spider silk hybrid particles are not immunogenic.

FIG. 2B shows the Cytokine quantification with ELISA. The adjuvant R848(R8), a TLR7 agonist, was used as positive control. This shows that thespider silk particles as well as the spider silk hybrid particles do nothave an intrinsic immunostimulatory activity.

Example 8: Spider Silk Particles are Efficiently Taken Up byAntigen-Presenting Cells

BMDC (5×10⁴ cells/well) were cultured with FITC-labelled spider silkparticles (C16-SIIN, C16-CathBseq-SIIN and C16-CathSseq-SIIN) at 50 ugparticle/mL according to example 6. After 24 hours of incubation, BMDCwere isolated for flow cytometry analysis.

The uptake of spider silk particles by BMDC was assessed by flowcytometry and confocal microscopy. For flow cytometry, dead cells wereexcluded using zombie violet dye (Biolegend, USA). After Fc receptorblocking, the following antibodies were added: APC-CD11b, APC-Cy7-CD11c(both from Biolegend, USA). Percentage of FITC+ positive cells in theCD11b+CD11c+ population was then determined. For confocal microscopy,BMDC were incubated with Blue DND-22 Lysotracker (Molecular Probes, USA)for 1 hour and FITC-positive particles for an additional 4 hours beforeimaging with confocal Microscopy (Zeiss, Germany). The percentage ofFITC-positive cells within BMDC (CD11c+) population was determined. SIINpolypeptide served as a negative control.

FIG. 3A shows the uptake of C16 particles without SIIN (C16), C16 hybridparticles C16-SIIN, C16 hybrid particles with Cathepsin B cleavage site(C16-CathBseq-SIIN) and C16 hybrid particles with Cathepsin S cleavagesite (C16-CathSseq-SIIN). Untreated cells (untreated) and SIINpolypeptide (SIIN) served as controls. A particle uptake of more than90% could be obtained for the hybrid particles. No significantdifference in uptake between the hybrid polypeptides C16-CathBseq-SIINand C16-CathSseq-SIIN and particles without antigen (C16) could bedetected. This shows that the hybrid particles are taken up as well asparticles without antigen (C16).

Splenocytes were isolated from C57BL/6JRj mice, passed through a 20 μmcell strainer and erythrocyte lysis was performed. Dead cells wereexcluded using violet zombie dye (Biolegend, USA). The freshly isolatedsplenocytes (5×104 cells/well) were cultured 6 hours with FITC-labelledspider silk particles. After Fc receptor blocking, the followingantibodies were added: PerCP-CD3, APC-CD11b, APC-Cy7-CD11c (all fromBiolegend, USA). After 6 hours of incubation, cells were analysed byflow cytometry.

FIG. 3B shows the percentage of FITC-positive cells determined indefined immune cell populations: T cells (CD3+), dendritic cells(CD11c+CD11b+) and monocytes/macrophages (CD11c-CD11b+) compared tountreated cells. The hybrid particles C16-CathSseq-SIIN were efficientlytaken up by dendritic cells, followed by monocytes/macrophages and Tcells.

Example 9: Cathepsin S Sequence is More Effective to Induce SIINFEKL(SEQ ID NO: 11) Dependent In Vitro T-Cell Proliferation

In Vitro T Cell Proliferation

BMDC (5×10⁴ cells/well) were cultured with spider silk particles at 50ug particle/mL. R848 (0.25 ug/mL) was used as adjuvant to induce BMDCactivation. After 24 hours of incubation, CFSE labelled CD3+CD8+ OT-Icells (105 cells/well) were added. After 3 days of co-culture, thedetection of CFSE positive CD3+CD8+ cells were analysed by flowcytometry.

After 24 h incubation, CD8+ T cells were negatively selected from OT-Isplenocytes using CD8+ T cell isolation kit (Miltenyi Biotech, Germany).These cells were then stained with CFSE (Molecular Probes, USA)according to manufacturer's protocol and added to the BMDC culture (10⁵cells/well). 3 days later, cells were stained for flow cytometryanalysis. After Fc blocking, the following antibodies were added:PB-CD3, APC-Cy7-CD8 (all from Biolegend, USA). The proliferation wasdetermined by the percentage of CFSE^(DIM) cells within the CD3+CD8+cell population.

FIG. 4A shows the percentage of proliferating CD8+ T-cells within the Tcell population (CD3+CD8+). The percentage of proliferating of CD8T-cells exposed to BMDC cells with C16 CathSseq-SIINFEKL particles andC16 CathBseq-SIINFEKL particles was significantly higher compared to theuntreated control. The percentage of proliferation of CD8 T-cellsexposed to BMDC cells with CathSseq-SIINFEKL was further significantlyhigher than the percentage of CD8 T-cells exposed to BMDC cells with C16CathBseq-SIINFEKL.

Hybrid protein particles are able to induce a T cell proliferation bythe release of the SIINFEKL (SEQ ID NO: 11) peptide. C16-CatS_SIINparticles are more effective than the C16-CatB-SIIN particles.

Example 10: Hybrid Spider Silk Particles Accumulate in the DrainingLymph Node In Vivo

For in vivo biodistribution studies, particles were injected in theflank of mice and 24 h later, the mice were sacrificed and organs wereharvested for analysis.

Female C57BL/6JRj (Janvier, France) and OVA-TCR transgenic OT-I mice(Charles River, Germany) were housed under specific pathogen-freeconditions and used at 6-12 weeks of age. Animal experimentation wasconducted according to the Swiss federal law for animal experimentation.

C57BL/6JRj mice were injected subcutaneously with 505 μg FITC-labelledC16-CathSseq-SIINFEKL particles in 100 μL PBS per mouse in the rightflank of 3 mice. PBS was used as negative control. After 24 hours, theipsilateral inguinal draining lymph nodes (DLN), the contralateralinguinal (non-DLN) and the spleen were isolated for flow cytometryanalysis and single cell suspensions were made as described above. Deadcells were excluded using violet zombie dye (Biolegend, USA). After Fcreceptor blocking, the following antibodies were added: PerCP-CD3,Pe-Cy7-CD11b, APC-CD11c (all from Biolegend, USA).

FIG. 5A shows the number of FITC positive cells (comprisingFITC-labelled C16-CathSseq-SIINFEKL particles) in draining lymph nodes(DLN) of 3 FITC-particle treated mice compared to a control group of 3PBS treated mice in several tissues (DLN, non-DLN and spleen.

The analysis showed that Cathepsin S linker particles(C16-CathSseq-SIINFEKL) accumulated in the local lymph nodes andpreferentially in dendritic cells and not in non DLN cells or spleen.During surgery for organ harvesting, particles were still at the placeof injection. The C16 hybrid particles are forming a depot under theskin and preferentially accumulate in the local lymph nodes.

FIG. 5B shows the uptake of Cathepsin S linker particles(C16-CathSseq-SIINFEKL) only in dendritic cells (CD11c+CD11b+) incontrast to monocytes/macrophages (CD11c-CD11b+). Dendritic cells arethe most potent class of antigen presenting cells. If presentation ofantigen happens at the local lymph nodes, particles do not diffuseuncontrollable through the body.

Example 11: SIINFEKL-Containing Spider Silk Particles InduceAntigen-Dependent T-Cell Proliferation In Vivo

In Vivo T-Cell Proliferation

After CD8+ T-cell isolation from OT-I splenocytes, the cells were thenstained with CFSE according to the manufacturer's protocol in theabsence of FCS. 10⁶ CFSE-labelled CD3+CD8+ OT-I cells in 100 μL of PBSwere injected intravenously into mice. 18 hours later, mice we in theright flank particles (in 100 μL of PBS) mice were immunizedsubcuteously with 505 μg C16 and C16-CathSseq-SIINFEKL particles (505 ugparticle in 100 μL PBS per mouse). R848 (25 ug in 100 μL) was used asadjuvant. PBS and R848 adjuvant without hybrid particles served as asnegative control. 3 days after vaccination, the inguinal DLN wereisolated for flow cytometry analysis to determine the proliferation ofCD3+CD8+ CFSE-labelled OT-I cells. Single cell suspensions were obtainedby passing through a 20 μm cell strainer. After Fc receptor blocking,the following antibodies were added: PB-CD3, APC-Cy7-CD8 (all fromBiolegend, USA). The proliferation was determined by the percentage ofCFSE^(DIM) cells within the CD3+CD8+ cell population.

Statistical Analysis

All graphs were made with GraphPad prism software version 6.0 g(GraphPad Software, San Diego, USA), where error bars indicate standarderror of means (SEM). Statistical significance of multiple groups tocontrol group (untreated sample) was performed using one-way ANOVAfollowed by Dunnett's multiple comparison test.

FIG. 6 shows the proliferation of CD8 T cells of mice immunized withC16-CathSseq-SIINFEKL particles. The proliferation of CD8 T cells wasremarkably higher without R848 adjuvant. It could be shown that theimmunization with C16-CathSseq-SIINFEKL without adjuvant resulted in asimilar respectively higher proliferation of CD8 T-cells than theimmunization with C16-CathSseq-SIINFEKL with adjuvant. This could not beexpected, because the immune response is usually higher after theco-administration of an immunostimulatory adjuvant.

The invention claimed is:
 1. A method for inducing an immune response ina subject comprising the steps of: (i) providing a polypeptidecomprising (a) a silk polypeptide, (b) an antigen, and (c) anenzymatically cleavable linker, wherein the antigen is connected to thesilk polypeptide via the enzymatically cleavable linker, and (ii)administering the polypeptide provided in (i) to a subject in needthereof.
 2. The method of claim 1, wherein the enzymatically cleavablelinker is a protease cleavable linker.
 3. The method of claim 2, whereinthe protease cleavable linker is a cathepsin cleavable linker.
 4. Themethod of claim 3, wherein the cathepsin cleavable linker is a cathepsinS cleavable linker.
 5. The method of claim 3, wherein the cathepsincleavable linker is a cathepsin B cleavable linker.
 6. The method ofclaim 1, wherein the polypeptide is comprised in a particle.
 7. Themethod of claim 1, wherein the polypeptide is comprised in apharmaceutical composition which is free of any adjuvant.
 8. The methodof claim 6, wherein the particle comprising the polypeptide is comprisedin a pharmaceutical composition which is free of any adjuvant.
 9. Themethod of claim 1, wherein the immune response is induced against theantigen in the patient.
 10. The method of claim 1, wherein the immuneresponse is induced against cancer, an infectious disease, or anautoimmune disease.
 11. The method of claim 1, wherein the induction ofthe immune response results in the immunization or vaccination of thepatient.
 12. A method for vaccinating a subject comprising the steps of:(i) providing a polypeptide comprising (a) a silk polypeptide, (b) anantigen, and (c) an enzymatically cleavable linker, wherein the antigenis connected to the silk polypeptide via the enzymatically cleavablelinker, and (ii) administering the polypeptide provided in (i) to asubject in need thereof.
 13. The method of claim 12, wherein thepolypeptide is comprised in a particle.
 14. A method for stimulating,priming, and/or expanding T cells in a subject comprising the steps of:(i) providing a polypeptide comprising (a) a silk polypeptide, (b) anantigen, and (c) an enzymatically cleavable linker, wherein the antigenis connected to the silk polypeptide via the enzymatically cleavablelinker, and (ii) administering the polypeptide provided in (i) to asubject in need thereof.
 15. The method of claim 14, wherein thepolypeptide is comprised in a particle.